Law enforcement officers are routinely exposed to hazardous, disturbing events that can impose severe stress and long-term psychological trauma. As a result, police and other public safety personnel (PSP) are at increased risk of developing posttraumatic stress injuries (PTSIs) and disruptions to the autonomic nervous system (ANS). ANS functioning can be objectively and noninvasively measured by heart rate (HR), heart rate variability (HRV), and respiratory sinus arrhythmia (RSA). Traditional interventions aimed at building resilience among PSP have not adequately addressed the physiological ANS dysregulations that lead to mental and physical health conditions, as well as burnout and fatigue following potential psychological trauma.In this study, we will investigate the efficacy of a web-based Autonomic Modulation Training (AMT) intervention on the following outcomes: (1) reducing self-reported symptoms of PTSI, (2) strengthening ANS physiological resilience and wellness capacity, and (3) exploring how sex and gender are related to baseline differences in psychological and biological PTSI symptoms and response to the AMT intervention.The study is comprised of 2 phases. Phase 1 involves the development of the web-based AMT intervention, which includes 1 session of baseline survey measures, 6 weekly sessions that integrate HRV biofeedback (HRVBF) training with meta-cognitive skill practice, and 1 session of follow-up survey measures. Phase 2 will use a cluster randomized control design to test the effectiveness of AMT on the following prepost outcomes: (1) self-report symptoms of PTSI and other wellness measures; (2) physiological indicators of health and resilience including resting HR, HRV, and RSA; and (3) the influence of sex and gender on other outcomes. Participants will be recruited for an 8-week study across Canada in rolling cohorts.The study received grant funding in March 2020 and ethics approval in February 2021. Due to delays related to COVID-19, phase 1 was completed in December 2022, and phase 2 pilot testing began in February 2023. Cohorts of 10 participants in the experimental (AMT) and control (prepost assessment only) groups will continue until a total of 250 participants are tested. Data collection from all phases is expected to conclude in December 2025 but may be extended until the intended sample size is reached. Quantitative analyses of psychological and physiological data will be conducted in conjunction with expert coinvestigators.There is an urgent need to provide police and PSP with effective training that improves physical and psychological functioning. Given that help-seeking for PTSI is reduced among these occupational groups, AMT is a promising intervention that can be completed in the privacy of one’s home. Importantly, AMT is a novel program that uniquely addresses the underlying physiological mechanisms that support resilience and wellness promotion and is tailored to the occupational demands of PSP.ClinicalTrials.gov NCT05521360; https://clinicaltrials.gov/ct2/show/NCT05521360PRR1-10.2196/33492 Law enforcement officers are routinely exposed to hazardous, disturbing events that can impose severe stress and long-term psychological trauma. Upward of 42% of public safety personnel (PSP) report one or more mental health symptoms . AccumulExisting PTSI prevention and resilience promotion interventions tailored to PSP not only show limited effectiveness but also do not consider sex and gender as central determinants of health and variables of importance ,9,10. FuAdvances in physiology and neuroscience demonstrate that resilience is maintained by healthy ANS functioning. Disruptions in ANS functioning can be observed by objectively and noninvasively measuring 2 biological indices ,14,16: hThere is evidence to suggest sex can influence ANS functioning, as indicated by differences in HRV and RSA at rest . These fResearch reveals a connection between respiration, arousal, and emotional control, such that how a person breathes sends signals to regions in the brainstem and forebrain that modulate arousal . For exaIn collaboration with expert police practitioners as well as medical and academic experts in psychophysiological risk and resilience, the lead author developed the International Performance Resilience and Efficiency Program (iPREP), an in-person resilience program that improves occupational health and performance by addressing the unique acute and chronic stressors faced by police -33. ThisThe aim of the proposed study is to develop (phase 1) and test (phase 2) if a web-based delivery of autonomic modulation training (AMT) can reduce symptoms of PTSI and build psychological wellness capacity and physiological resilience in police officers with similar effectiveness as in-person training. An additional novel scientific contribution of this protocol includes an examination of sex and gender in baseline biological and psychological presentations of PTSI among police and in response to a resilience-building intervention.The following 3 hypotheses were proposed:H1: Symptoms of PTSI will be significantly reduced following the completion of the AMT intervention.H2: Functional wellness capacity and resilience will be significantly improved following the completion of the AMT intervention.H3: Sex and gender differences in PTSI will be observed at baseline and postintervention assessments.Specifically, we expect that self-identified females will report more symptoms of PTSI and lower resilience at baseline, a greater reduction of PTSI symptoms, and a greater increase in resilience at follow-up than self-identified males. Gender role stress, discrimination, and harassment will be explored as moderators of PTSI symptoms at baseline and in response to the AMT intervention.The following 4 hypotheses were proposed:H4: Resting HRV will significantly increase following the completion of the AMT intervention.H5: RSA will significantly increase following the completion of the AMT intervention.H6: Recovery from acute stress will significantly improve following the completion of the AMT intervention.H7: Based on inconsistencies in the extant literature, we will conduct exploratory analyses of any potential sex and gender differences in HRV, RSA, and acute stress recovery at baseline and postintervention assessments.This protocol has been approved by the University of Toronto’s Social Sciences, Humanities, and Education Research Ethics Board (Protocol #39478).Capitalizing on the numerous long-standing professional relationships between study coinvestigators and police populations of interest, phase 1—development of the AMT intervention—will use an integrated knowledge translation (iKT) approach to generate user-informed content . To testWe aim to test the AMT intervention on a sample of approximately 250 frontline police officers in rolling cohorts of 10-15 participants  from geographically representative police services across Canada. A power analysis was conducted using G*Power software  and founParticipants will be recruited in several ways: (1) partnering police services will provide a link to the web-based study hosted on the confidential University of Toronto  servers to members via email, announcements, and internal media postings with encouragement to participate; (2) the research team will visit police services and other related fora  to hold recruitment and information days where eligible officers may sign up for the study; (3) our knowledge user (MLV) and expert police practitioner collaborator (PS) will facilitate recruitment of law enforcement officers through targeted media attention, networking, and professional outreach activities; (4) supporting stakeholder groups will facilitate communication and recruitment with users and law enforcement officers. Based on delays due to the COVID-19 pandemic, the order in which partner organizations are recruited and tested is subject to change based on availability and current public health guidelines.Interested participants will be directed to a web-based consent form on a secure site that will detail the study aims, eligibility criteria, intervention protocol, allocation and procedures for experimental and control groups, risks and benefits, and other information related to their rights as a research participant . Participants will provide their informed consent by clicking on a link that will take them to an eligibility screening survey that will collect the following identifiable information: full name, mailing address (for equipment shipping and incentive payment), personal email address (for correspondence with the research team that is not monitored or accessible by the participants’ police agency), and demographics including sex at birth, gender, age, ethnicity, police service (to verify active duty status against publicly available directories), role, years of service as a police officer, and confirmation that they are not currently on extended medical leave. Participants deemed eligible for the study will be provided with deidentified usernames and passwords to access the preintervention assessment (see phase 2 procedures).Inclusion criteria included adults (aged 18 years or older) currently employed as active duty  frontline law enforcement officers fluent in English. Exclusion criteria include non-Canadian law enforcement officers, police administrators, civilian employees , and officers that do not have regular access to a computer with internet access to complete the web-based AMT intervention. Scores on self-reported mental health screening tools completed during the preintervention sessions will be used to match the assignment of participants to experimental or control groups but will not be used to exclude participants based on established cutoff scores for high or severe PTSI symptoms.Using an iKT approach, the multidisciplinary research team will consult with police practitioners to tailor AMT content  and promote participation and adherence to the AMT intervention by advising on procedural aspects of the research study . The AMT intervention is comprised of 2 components—HRVBF training and meta-cognitive coping skills.Based on an established and publicly available protocol , HRVBF hIntegrated with HRVBF training, psychoeducational modules will be developed to help individuals cultivate meta-cognitive coping skills, defined as the awareness and understanding of one’s own thought processes and learning how to manage the assessment of one’s own experiences, correctly identify demands and resources, and regulate the appraisal of experiences using attentional and cognitive techniques . ParticiResearch ethics approvals, data sharing agreements with partner institutions, and the procurement of equipment to record HRV and HRVBF will also be finalized during this phase of the study.The ention assessment, 6 weeks of AMT sessions, and a postintervention assessment. Once participants are deemed eligible for the study, they will be issued a unique deidentified username to access the web-based study, which will be hosted on secure LMS and survey platforms .During this session participants will complete self-reported baseline measures of: (1) PTSI, including the Personal Health Questionnaire (PHQ-8) for major depressive disorder, General Anxiety Disorder Scale (GAD-7), Depression, Anxiety, and Stress Scale (DASS-42), PTSD checklist (PCL-5), Alcohol Use Disorder Identification Test (AUDIT), Perceived Stress Scale (PSS), and Personal Burnout Subscale of the Copenhagen Burnout Inventory (CBI) -71; (2) Upon completion of session 1, participants will be asked if they want to continue in the study. This secondary “reconsent” is designed to conserve shipment of expensive study equipment  and retain eligible participants that demonstrate commitment to adhering to the study protocol. If participants agree to continue, they will receive a notification in their personal email confirming their mailing address and will be shipped necessary study materials, including compensation for completing session 1, a wearable HR monitor and chest band, a device equipped with the HRVBF app (an iPad), and a prepaid envelope to return all equipment at the end of the study. Once participants confirm receipt of their equipment in the mail, they will gain access to session 2.All participants will be taught via web-based video modules how to apply the wearable HR monitor and configure the HRVBF app. The wearable HR monitor will automatically record date and time-stamped physiological data upon contact with the skin via the chest band and will communicate with the HRVBF app via Bluetooth. All HRV data will be stored locally on the HRVBF app and study device, which will be mailed back to the research team at the end of the study period. In the first part of this session, both experimental and control participants will provide baseline resting HRV and RSA data that will be recorded while participants engage in a 7-minute neutral activity  that does not stimulate arousal or relaxation . To assess recovery from an acute stressor, participants will perform web-based versions of the Paced Auditory Serial Addition Test (PASAT)  and the Following these baseline physiological assessments, participants in the experimental group will continue with the rest of their session (15-20 min) and begin the AMT intervention. Participants in the control group will not have any programming until session 8 (postintervention assessment).These sessions will be completed by participants in the experimental group only. The content of each AMT session will be modified from the Lehrer et al  HRVBF prAll participants will receive an email reminder 1 week before the final session, which will be completed by both experimental and control participants. It will begin by obtaining objective physiological measures at rest, including HRV , RSA (paced breathing), and recovery from acute stress (PASAT and Stroop tasks). Next, participants will complete the same self-reported questionnaires as in session 1 to assess changes in self-reported PTSI, resilience and well-being, occupational and operational stress exposure, and sex and gender-relevant data (see hypotheses). Participants in the control group will be asked if they would like to complete the AMT intervention. If so, they will be provided access to sessions 3 to 7 and will be asked to complete a second postintervention assessment (session 8) after completing the AMT intervention. Incentive payments will be processed as participants drop out or complete study assessments.Following cohort 1, we will invite participants to take part in iKT Focus Groups to generate user-informed feedback on the web-based AMT intervention and identify potential sex and gender differences in the user experience. Focus groups will be led by our sex and gender expert (RR) and use a semistructured, purposive, snowball sampling design. Half-day focus groups will be held in person, with dedicated focus groups for female and male participants to provide sex-stratified samples. We will limit focus groups to 5 participants, as previous research shows groups of 4-6 ameliorate the effects of “over sharing” or domineering participants, as well as participants who may feel intimidated and become silent within larger groups ,90. GrouIn accordance with our hypotheses, PTSI (H1) will be measured by the PHQ-8, GAD-7, DASS-42, PCL-5, AUDIT, PSS, CBI, and self-reported stress visual analog scale. Self-reported resilience and well-being (H2) will be measured by the Brief COPE, RSA-33, BRS, WBSI, PTQ, UB-PSWQ, and UB-RRS. Functional wellness capacity and occupational stress will be measured by the SDS, GHPS, COVID-19 HRSI, WHODAS 2.0, PSQ, and LEAPS. Sex will be measured by self-reported sex at birth ; gender is measured by self-identification as male, female, nonbinary, or other (with the option to self-report) or “prefer not to disclose.” Sex and gender-related experiences will be measured by the WGDS and GEQ (H3 and H7). Changes in resting HRV (H4) will be measured by time and frequency domain parameters based on data collected from the wearable HR monitors  as recommended in the standards of practice for HRV analysis . RSA , and will not be shared with collaborators or participating police organizations. Data collected on the LMS and secure survey platforms  will be matched to a deidentified user ID. Physiological data gathered by the wearable HR monitor and HRVBF app will be stored locally and will not be transferred to publicly accessible third-party cloud-based servers. Rather, participants will mail the equipment back to the research team, which will match the deidentified data offline. The key code linking participants’ personal information and deidentified user IDs will only be stored on secure servers at the University of Toronto and will be destroyed once the participant completes or withdraws from the study, has received their compensation, and all deidentified data files have been matched.Compute Ontario Advanced Research Computing equipment at the Centre for Advanced Computing (Queen’s University). CM will design the data management, cleaning, and analysis protocols necessary to construct the big data pipeline to process the large amount of continuous data, including matching the time stamps on the physiological data to significant event markers in the AMT intervention  and on the HRVBF app . Preprocessed data will then be sent back to the primary research team  via a secure VPN tunnel and housed on the secure internal University of Toronto server (RES-NAS) for the statistical analyses described below using bespoke syntax and code on various software platforms . Data from iKT focus groups will be stored in RR’s secure data web-based Al Fresco space and hard copies in her triple-locking filing cabinet in her office at the Memorial University of Newfoundland.Physiological data will be transferred to a 2-time Canada Research Chair in Health Informatics and Big Data expert coinvestigator (CM) via a secure virtual private network (VPN) tunnel and housed on a private cloud-based Big Data analytics platform located within the To test the effectiveness of the AMT intervention on reducing PTSI symptoms and improving resilience, we will conduct prepost analyses of the self-reported PTSI (H1) and resilience measures (H2) using separate repeated measures analyses of covariance (ANCOVAs) accounting for demographic covariates for normally distributed data, and ranked repeated measures ANCOVAs for nonnormally distributed data . VariablTo test the effectiveness of the AMT intervention on improving biological indicators of resilience, prepost analyses of resting HRV parameters (H4), RSA (H5), and recovery from acute stress (H6) will be separately assessed using repeated measures ANCOVAs with sex, gender (H7), age, years of service, and geographic service region as covariates. To determine which factors significantly predict greater changes in biological outcome measures, hierarchical linear regression analyses will be undertaken with self-reported measures and demographic variables entered as predictors in the models. We will also compare weekly changes in HRV and RSA as measured during AMT module exercises with repeated measures ANCOVAs. A main effects analysis of the “time” variable will explore the potential incremental benefit of AMT training and identify at what point during the 6-week intervention significant biological changes occur. Computational approaches to analyzing and visualizing biological indices of resilience will be led by CM, including integration of cardiac  and respiratory  rhythms to elucidate patterns of desynchronization before, during, and following AMT.To explore sex and gender-based contributions to self-report (H3) and objective indices (H7) of PTSI and resilience, the above analyses will include sex and gender as covariates or predictor variables with sex-stratified samples. These findings will clarify any sex-related differences in baseline and postintervention rates of PTSI and resilience among law enforcement officers and whether there are any sex-related differences in the magnitude of treatment effects (H3). Moderated regression analyses will test whether the AMT intervention affects the relationship between sex and PTSI symptoms and resilience. Multiple comparisons will be adjusted with Bonferroni corrections, where applicable.Qualitative analyses of the transcribed data obtained from the iKT focus groups will be led by the sex and gender coinvestigator (RR) with the assistance of graduate students and postdoctoral fellows, with the aim of identifying sex- and gender-based considerations for developing effective occupational health interventions tailored to police officers and other PSP groups. Qualitative data analysis will use an inductive, comparative approach without initially arriving at any definitive, substantive, or theoretical conclusions about what the data reflect interpretatively with a lens on sociology and criminology . Themes The study received notice of funding acceptance in March 2020. In February 2021, ethics approval was obtained from the University of Toronto’s Social Sciences, Humanities, and Education Research Ethics Board (Protocol #39478) and was renewed in February 2023. Phase 1 of the study (AMT Development) was completed in December 2022. Participant recruitment for phase 2 began with the pilot cohort in February 2023 via advertisements through partnering stakeholders (see Recruitment section). As of April 2023, 7 pilot participants were actively registered for the AMT intervention. Knowledge dissemination for phase 1 was completed in March 2023 at an annual meeting of the public safety wellness team grant holders and is planned for 1 scientific (May 2023) and 1 practitioner (October 2023) conference. iKT focus groups will take place following the completion of each sufficient cohort (see the iKT Focus Groups section above).Based on the extant and precedent literature, we predict that the stated hypotheses will be confirmed following the execution of the proposed protocol. Specifically, we anticipate significant reductions in PTSI symptoms and improvements in physiological indicators of health and resilience following the completion of the 6-week web-based AMT intervention. We also anticipate sex- and gender-based differences in pre- and postintervention outcomes, such that self-identified females will report higher PTSI symptoms and lower resilience at baseline and a greater reduction of PTSI symptoms and greater increase in resilience at follow-up than self-identified males. The direction and magnitude of sex- and gender-based differences in physiological outcomes are not currently specified, given the novelty of these analyses. Insights derived from iKT focus groups will supplement the quantitative sex- and gender-based analyses (H3 and H7).AMT fills an existing gap in the applied research literature supporting evidence-informed prevention and intervention programs for PSP exposed to PTSI, such that AMT aims to measure and strengthen objective biological resilience . SpecifiAMT techniques have previously garnered high “buy-in” from participating police officers because the intervention provides tangible, real-time, biological evidence of occupational and health improvement from the very first HRVBF session -34. Buy-Pending significant results, AMT can be delivered alone or in tandem with other psychotherapeutic treatments, such as cognitive behavioral therapy, to provide PSP services with an attractive suite of resilience and wellness capacity interventions that reduce PTSI symptoms and save operating costs  . The intRapid and direct knowledge dissemination to various stakeholders  will be facilitated by knowledge users’ (MLV and PS) capacities as retired PSP, board members, and English-French bilingual contributors to police periodicals including “The Blue Line.” In addition to this protocol, several research articles will be prepared based on the planned objectives and analyses identified above. High-impact peer-reviewed journals in relevant fields  that provide open access options will be prioritized. Research findings on the pilot (n=30) and complete data sets (n=250) will be disseminated at scientific  and applied practitioner conferences  to distribute new knowledge generated to the widest audience possible.


Twice-a-year, large-scale movement of billions of birds across latitudinal gradients is one of the most fascinating behavioral phenomena seen among animals. These seasonal voyages in autumn southwards and in spring northwards occur within a discrete time window and, as part of an overall annual itinerary, involve close interaction of the endogenous rhythm at several levels with prevailing photoperiod and temperature. The overall success of seasonal migrations thus depends on their close coupling with the other annual sub-cycles, namely those of the breeding, post-breeding recovery, molt and non-migratory periods. There are striking alterations in the daily behavior and physiology with the onset and end of the migratory period, as shown by the phase inversions in behavioral  and neural activities. Interestingly, there are also differences in the behavior, physiology and regulatory strategies between autumn and spring  migrations. Concurrent molecular changes occur in regulatory (brain) and metabolic  tissues, as shown in the expression of genes particularly associated with 24 h timekeeping, fat accumulation and the overall metabolism. Here, we present insights into the genetic basis of migratory behavior based on studies using both candidate and global gene expression approaches in passerine migrants, with special reference to Palearctic-Indian migratory blackheaded and redheaded buntings. Avian migration is a regular, seasonal, large-scale movement of a population between fixed breeding and wintering geographical locations . BillionThree experimental approaches have been generally used to study the genetic control of migratory behavior in birds . These aA successful migratory journey requires the occurrence of sequential changes at several levels with great precision. It is therefore important that a migratory trait has been genetically shaped and adapted to the external environment. Over the years, this proposition has been experimentally tested, as described briefly below.Sylvia atricapilla); ~40% of F1 hybrids were migratory [Early in the 20th century, the experimental evidence came for an innate nature and inheritance of avian migratory behavior ,10,11. Aigratory ,12. Mostigratory ,12. Furtigratory  and direigratory .Cuculus canorus). Satellite tracking of migrating cuckoos revealed that juveniles had initiated migratory travel later than the adults, and they traveled via a straighter and faster route to reach their African wintering grounds, perhaps as independently guided by their innate migration programs [There is also good evidence to show that a migrant can complete its migratory travel without guidance from experienced conspecifics, as concluded from a study on common cuckoos (programs .Sylvia borin) was fairly insensitive to food availability or weather conditions, although a very long photoperiod could modify the amount of night activity [Phylloscopus trochilus) subspecies that have drastically different migratory routes. The subspecies P. t. trochilus migrates southwest from western European breeding to western African breeding grounds, while P. t. acredula migrates from southeast from northern and eastern Europe to eastern and southern Africa [P. t. trochilus was associated with the trochilus allele at inversion polymorphisms on chromosomes 1 (InvP-Ch1), and to southeastern migratory direction in P. t. acredula was associated with migration associated repeat block (MARB-a); MARB-a seemed to have an epistatic suppressive effect on the InvP-Ch1 [Both spatiotemporal migration program and migratory activity are heritable ,12,16. Aactivity ,19,20. Ln Africa ,22. The n Africa . Very ren Africa  discoverInvP-Ch1 .Acrocephalus arundinaceus). There was 16.4% heritability for the arrival dates as well as directional selection for early arrivals in both sexes acting through the reproductive success [An important aspect of migration is the timing of arrival at the breeding site in order to have prior access to the prime breeding habitat and a better mate choice . A decli success .Studies over the last three decades also suggest a correlation of rising mean spring temperature with the arrival date and hence the early start of reproduction . Given tA comparative study of gene polymorphism in an experimentally simulated  or in naturally occurring  functional states can reveal the genetic basis of a trait, such as seasonal migration.Adcyap1 (adenylate cyclase activating polypeptide 1) gene polymorphism with the migratory restlessness in blackcaps; birds with longer Adcyap1 allele exhibited higher Zugunruhe [CLOCK (circadian locomotor output kaput) gene polymorphism was found associated with enroute stay (stopover) times  in trans-Saharan migratory common nightingales (Luscinia megarhynchos), European pied flycatchers (Ficedula hypoleuca), tree pipits  and whinchats (Saxicola rubetra) [Adcyap1 or CLOCK gene allele lengths with the migratory status in a study of 15 different populations across two subspecies of Junco ranging from sedentary to long-distance migrants. Notably, however, the long-distance migratory Juncos had longer CLOCK alleles on average and, similar to captive blackcaps, the Adcyap1 allele length showed a positive correlation with the migratory restlessness among individuals of one of the two Junco populations studied, indicating the association of Adcyap1 gene with migratory propensity within or between certain populations only [CLOCK gene polymorphism was also not associated with the migration phenotype in bar-tailed godwit (Limosa lapponica baueri) [Using gene polymorphism as a molecular approach, few studies have documented the association of a single gene polymorphism with the migratory phenotype, while few others have failed to show such an association. For example, there was a significant correlation of ugunruhe . A similrubetra) ,38. At trubetra)  found no baueri) . Vps13a (vacuolar protein sorting 13 homolog A) gene was found to be associated with migration directionality in two very closely genetically related Vermivora warbler species with similar breeding sites in North America and vastly different wintering sites in different geographical directions—golden-winged warbler (Vermivora chrysoptera) winters in Central America and blue-winged warbler (V. cyanoptera) winters in South America [Vps13a gene region among South America wintering warblers, indicating the likelihood of natural selection on this gene locus [Falco peregrinus) indicated the association of Adcy8 (adenylate cyclase 8) gene with population level differences in the migratory distance [Similarly, a single nucleotide polymorphism of  America ,42. Therne locus . Likewisdistance . Falco sparverius) further suggests this [Top1, Phlpp1, Cpne4 and Peak1 genes) accounted for an intra-population existence of both early and late migratory chronotypes, supporting the argument that the variation in regulatory gene regions was responsible for the variation in migration phenotypes [Notably, blackcaps have emerged as an experimental model system to study the genetics and epigenetics of migration . To tracsts this . In kestenotypes .A migratory phenotype is expressed for a specific purpose and for a defined time period. Additionally, the two seasonal migrations (to-and-fro movement between breeding and wintering grounds) differ in several ways. For example, spring and autumn migrations differ in context: the spring travel is for the timely arrival at the breeding grounds (hence there is a stronger reproductive drive), whilst the autumn travel is essentially for escaping harsh winter conditions at breeding grounds and for finding adequate food resources for spending the winter season. This is reflected in the higher speed, longer nocturnal flights and shorter stopovers during spring than during the autumn migration ,49,50. BEmberiza melanocephala and redheaded bunting, Emberiza bruniceps. Some of the results are described in the following headings.In recent years, several gene expression studies have been conducted in long distance Palearctic—Indian migratory emberizid finches, the blackheaded bunting, Dio2, a gene that encodes for type 2 deiodinase, DIO2, the thyroid hormone activating enzyme via the Tsh-receptor-cAMP signaling pathway [The annual change in the relative length of daily light and dark periods away from the equator or in the relative intensity of daytime intensity around the equator provides a robust time cue for appropriate timing of behavior and physiology during the year ,58. The  pathway . DIO2 ca pathway . Pax6 (paired box 6) involved in the activation of Eya3 , Tshβ, Dio2 and Rhodopsin genes [Tshβ and Dio2 and a decrease in Dio3 level was found by hour 18 of the first long day in migratory blackheaded bunting . In an initial experiment on migratory blackheaded bunting in which birds were exposed for four weeks to near-threshold photoperiods at an ambient temperature of 22 °C and 35 °C, there was a reduced body fattening, attenuated activity level and delayed onset of Zugunruhe at the higher temperature [Tshb-Dio2-Gnrh gene pathway was upregulated at a high temperature (38 °C), while expression of the Trpm8 gene  was found to be high at 22 °C, compared to 38 °C  were upregulated at 35 °C . In a sBirds accumulate fat (fuel), which is drastic even in small birds and in those that are principally nocturnal migrants . In factThe fat deposition  is largely by hyperphagia, for which both physiological and molecular underpinnings are largely unknown. At this time, the best studied approach with respect to fueling is via the unraveling of mechanisms involved in hunger and feeding, i.e., food intake, which to a large extent is influenced by the energy status and nutrient availability. The hypothalamic arcuate nucleus plays a critical role via homeostatic balance of the activity of orexigenic  and anorexigenic  neurons .Npy in the hypothalamus showed seasonal alterations, with a loss of a significant daily rhythm in the photoperiod-induced migratory state. The high amplitude Npy daily rhythm during the winter non-migratory state with peak lying at the end of the dark phase possibly indicated a long-night starvation under winter short days [In a study related to changes in brain peptides associated with energy homeostasis, Surbhi et al.  found seort days .A closely related aspect of migration is how migrants decide that they are ready to continue their journey, i.e., stays at the stopover sites. In a study, Goymann et al.  studied circa = about, dies = day) rhythm [Zugunruhe in captive migratory birds [Endogenous clocks enable a long-lived species to identify the time of the year to switch on and switch off its internal mechanisms, in order to synchronize behavior and physiology with the favorable season. In a photoperiodic species, the internal clock is sensitive to daily changes in the photoperiod. The photoperiodic clock shows features that are consistent with a circadian  rhythm . Each da) rhythm , and resry birds . This seZugunruhe. When Zugunruhe is expressed, the free-running period of locomotor activity lengthens [Zugunruhe and diurnal hopping and feeding behaviors in migratory white-throated sparrows [Zugunruhe but decayed the circadian rhythm in Zugunruhe as well as in clock genes  in the hypothalamus, but not in the retina [Rorα and RevErbα gene expressions were phased inversed in parallel with the changes in the activity behavior with the development of Zugunruhe in blackheaded buntings.An endogenous circadian clock controls the daily alteration between the diurnal hopping and feeding behaviors and the nocturnal appearance of engthens ,82. The sparrows . In migre retina . InteresExperiments on migratory blackheaded bunting have also shown the alteration in daily waveform of clock gene oscillations in both central  and peripheral (muscle and liver) tissues . There wTh gene in migratory redheaded buntings with simulated spring migration phenotype, as compared to those with non-migratory as well as the autumn migratory phenotype , compared to when they were exposed to a longer light wavelength (620 nm) [Th mRNA expression, compared to levels at 22 °C, in both the midbrain and hypothalamus of migratory blackheaded buntings  also showed differences in their expression patterns, with higher Dio2 and Dio3 mRNA levels in buntings exhibiting spring and autumn migratory phenotypes, respectively [As noted above, the spring and autumn migratory travels are distinctly different, and so they probably need differential molecular strategies to achieve their goals. In a series of investigations, Kumar and colleagues investigated this mainly using redheaded buntings as the experimental system . In  pathway . The thyectively .Atgl) gene [Fat/Cd36), fatty acid binding protein (Fabp3) and carnitine palmitoyl transferase (Cpt1) genes, which are responsible for drawing free fatty acid from the circulation into flight muscle for its β-oxidation and hence energy generation, were also higher in birds in the spring migratory than the autumn migratory state. Increased muscular expression of Myod1 and Pvalb genes further suggested an enhanced myogenesis, hence an increased support to the strength of extensively working flight muscles in spring migration , buntings exhibiting spring migratory phenotype showed a higher mRNA expression of genes involved in the fat metabolism . For exagl) gene , which igl) gene . The mRNMigration is not a single component event; it is rather an outcome of several sequentially occurring component events, viz., the migratory urge, preparedness for the long journey, direction to fly, distance to cover and the mechanism to replenish energy during stopovers. Therefore, instead of a single or a set of genes, the genetic control for migration may involve an entire “migratory gene package”. The constituent genes of such a “gene package” must show differential expression possibly with varying degrees among its candidates during different stages of the migration. The study of such a proposition has become possible through recent technological advancements, such as the next-gen sequencing technique that allows for whole-genome or transcriptome sequencing.P. t. trochilus and P. t. acredula and found 55 highly differentiated SNPs between two subspecies clustering largely to two chromosome regions, possibly influenced by the divergent selection and adaptation to differential migratory strategies [Junco hyemalis). There was an increased expression of genes associated with lipid transport and fatty acid catabolic processes in the muscle and with a ribosomal structure that indicated protein synthesis in the blood of the migratory J. h. hyemalis, as compared to those in the sedentary J. h. carolinensis [Using the global gene approach, there have been attempts in the last ten years to identify the component genes of the “migratory gene package”. In a first study of the kind, using 454 pyrosequencing, Lundberg et al.  comparedrategies . Similarrategies  found 54linensis .Catharus ustulatus). On the other hand, Franchini et al. [Turdus merula).Further studies focused on the differences in gene expressions in between seasonal states of the same species. Using microarrays, Boss et al.  reportedi et al.  reportedMore recently, our laboratory has performed a number of transcriptome analyses of the hypothalamus and liver tissues of migratory blackheaded and redheaded buntings. We found an enhanced expression of genes enriching the ATP binding pathway, focal adhesion and intracellular protein transport in the hypothalamus and that of calcium ion transport and small GTPase-mediated signal transduction in the liver of blackheaded buntings exhibiting a photostimulated spring migration state . The RNAThe present review is an update on advances in the genetic control of seasonal migration in birds, with a particular focus on latitudinal passerine migrants that outnumber the other groups of avian migrants. It has to be considered that different genetic programs might have evolved to anticipate and tune with several changes in the external environment (abiotic factors) that create a short favorable window and shape the execution of a high-energy seasonal event such as migration. Although the primary abiotic driver of seasonal migration is the change in environmental photoperiod , there aThe internal genetic programs involve a host of cellular signaling cascades and operate in sync with each other so that they can effectively integrate and transduce information from the environment to relevant effector organs which are functionally ready when they are needed. For example, the mechanisms underlying timekeeping will create the temporal window  andWe believe that the recent global genetic approach is significantly advancing our knowledge of candidate molecules that possibly underpin the seasonal biological processes, such as migration in birds. However, there is a need for more intense and concerted research on (i) the characterization and differential expression of a host of genes at different stages of annual life history, and (ii) how differentially expressed candidate genes are translated and subjected to post-translational modifications that ultimately govern the biological function. We would like to note further that (i) within and between population genetic variations in migratory traits might not be found in the sequence of differentially expressed genes, but rather in regulatory elements upstream or elsewhere in the genome , and (ii


Optical microscopy enables the non-invasive investigation of the dynamic structure and function of living organisms, providing the necessary spatial resolution to resolve cellular and subcellular components. In neuroscience, this unique ability has been translated into imaging the activity and connections of individual neurons and their processes in the living brain. The highly heterogeneous nature of biological tissues, however, causes refractive aberrations to the light used by optical microscopes, compromising the image brightness and resolution and limiting the imaging depth, ultimately holding back our understanding of many biological phenomena of interest. To address the challenge associated with imaging heterogeneous tissues, adaptive optics actively shapes the wavefront of light and compensates for the optical aberrations introduced by biological specimens, enabling subcellular resolution imaging at depth Booth, . This ReHung et al., the authors perform 3D single-molecule localization microscopy (SMLM) in tissues by combining adaptive optics (AO), in situ 3D point spread function (PSF) calibration, and 3D single objective lens inclined light sheet (SOLEIL) microscopy  using an azimuthally polarized vortex excitation beam and (2) modulating the amplitude distribution of the azimuthally polarized vortex beam using a multi-ring mask  fluorescence microscopy (2PFM) dramatically extends the imaging depth in tissues—when compared with one-photon-fluorescence-based techniques that use visible light for fluorescence excitation—and achieves optical sectioning due to the nonlinear excitation confinement , as expected given the significant additional aberrations added by the overlaying layer of white matter.As the imaging depth is increased in tissues, the 2P excitation power needs to be increased exponentially to compensate for the scattered photons. As the power continues to be raised, the nonlinear excitation will no longer be confined to the focal region and the out-of-focus signal will approach that generated within the focal volume—at which point the maximum imaging depth for 2PFM is reached. Owing to the tighter signal confinement provided by the higher-order nonlinear excitation, and the reduced scattering experienced by longer excitation wavelengths, three-photon fluorescence microscopy (3PFM) extends the penetration depth in tissues beyond the limits of 2PFM (Wang and Xu, Sohmen et al., the authors discuss how turbidity can be quantified, and device a numerical model to emulate the effect of a 3D scattering medium of “tunable” transport mean free path on a light field by using a 2D phase mask displayed on an SLM. Using this model as well as experiments, the authors investigate the performance of a recently introduced approach called DASH (May, To correct for wavefront distortions outside of the traditional regime dealing with lower-order wavefront aberrations, different methods can be applied. In the work by ASH May, —Dynamic ASH May,  which thThe contributions in this special issue highlight the important role that adaptive optical methods continue to play in a range of microscopy modalities including super-resolution and multiphoton microscopy. By canceling out the aberrations introduced by inhomogeneous tissues, such as the brain, adaptive optical methods improve imaging performance and enable high resolution imaging of tissues at depth. We envision aberration correction modules will become an indispensable add-on component to optical microscopes in many laboratories, opening new avenues for biological exploration. To this end, continued efforts to make such methods readily available and easy to implement to non-experts will be essential.RT was the overall editor of the Research Topic. CR wrote the editorial. All authors checked and confirmed the final version of the editorial. All authors contributed to the article and approved the submitted version.


Resistance to drug therapy is a major hurdle in new-onset refractory status epilepticus (NORSE) treatment and there is urgent need to develop new treatment approaches. Non-drug approaches such as neuromodulation offer significant benefits and should be investigated as new adjunct treatment modalities. An important unanswered question is whether desynchronizing networks by vagal nerve stimulation (VNS) may improve seizure control in NORSE patients.We present a summary of published NORSE cases treated with VNS and our own data, discuss possible mechanisms of action, review VNS implantation timing, stimulation setting titration protocols and outcomes. Further, we propose avenues for future research.We advocate for consideration of VNS for NORSE both in early and late stages of the presentation and hypothesize a possible additional benefit from implantation in the acute phase of the disease. This should be pursued in the context of a clinical trial, harmonizing inclusion criteria, accuracy of documentation and treatment protocols. A study planned within our UK-wide NORSE-UK network will answer the question if VNS may confer benefits in aborting unremitting status epilepticus, modulate ictogenesis and reduce long-term chronic seizure burden. New-onset refractory status epilepticus (NORSE) and its subcategory febrile infection-related epilepsy syndrome (FIRES) are rare, devastating clinical presentations with <500 cases reported in the literature to date , 2. TheyThe anti-ictal and anti-epileptogenic mechanisms of action of VNS have been studied extensively in both humans and animal models and comprise stimulation of serotonergic and noradrenergic centers in the brainstem , norepinClinicalTrials.gov, and PubMed databases using the following search strategy , in the chronic phase of treatment-resistant epilepsy (TRE) in seven cases  and had failed multiple standard antiseizure medications, anesthetic agents and trials of immunosuppression . Our patient had also undergone an unsuccessful trial of electroconvulsive therapy and repetitive transcranial magnetic stimulation. VNS was switched on immediately after insertion, initial stimulation started with 0.5 mA output current, and gradually increased to 2 mA, 30 Hz, 500 μS, with a duty cycle of 35% (30 s ON and 1.1 min OFF). Case Ictal activity on EEG resolved on day 2 after implantation, allowing gradual tapering of clonazepam and anesthetic agents, and leaving the ICU. She died 46 days after VNS implantation due to an obstructed tracheostomy and cardiac arrest.Case 2—Early implantation of VNS in acute phase of NORSEin pregnancyA 30-year old pregnant female in the first Trimester of pregnancy was implanted with VNS in the acute phase (day 26 from onset) of NORSE possibly linked with drug overdose. She had also failed multiple standard antiseizure medications, anesthetic agents and trials of immunosuppression including Anakinra. VNS was switched on the day of implantation with initial output current of 0.25 mA. Output current was further uptitrated to 1 mA in the following 72 h, and increased to 1.25 mA on day 7 post-op. Our patient experienced improvement of myoclonic jerks from day 7 post-implantation and became seizure-free from day 20 post implantation. She regained functional independence during inpatient rehabilitaton, delivered a premature but healthy baby at 33 weeks and has remained seizure free to date (last reviewed 8 months from onset).Studying rare and complex diseases such as NORSE in the real clinical world is challenging, as patients may be subject to multiple and concomitant interventions and the presence of publication bias toward cases with good outcomes is very likely. Due to the paucity of cases and the variable amount of information available within each report, the level of evidence supporting the use of VNS in NORSE is low. Nevertheless, we feel that from the cases summarized in the previous sections and our experience, some general conclusions can be drawn: overall, VNS was a well-tolerated intervention without significant adverse effects in the short or long term, both in cases implanted acutely or in the TRE phase, supporting its safety even in pregnancy. Whilst it is not possible to determine a stimulation threshold effect leading to seizure cessation, most patients had VNS switched on either immediately or within the first few weeks of implantation at conventional—not high frequency—cycling rates and the output current increased over a short period of time (days to weeks). In the three cases, including ours , 23, wheThe original contributions presented in the study are included in the article/LMR and RS designed the work. LMR wrote the manuscript. Both authors revised the manuscript, read and approved the final version.


Accumulated evidence has shown that CKD increases the risk of cardiovascular diseases (CVD) such as myocardial infarction, stroke, and heart failure (HF), as well as death , and CKD should be an important target for primary CVD prevention such as heart failure (6).Chronic kidney disease (CKD) is a clinical diagnostic definition that comprehensively expresses pathological conditions where kidney damage or decreased kidney function persists (8). In addition, a large-scale cohort study of patients with CKD in Japan has reported that HF is more frequent than myocardial infarction and cerebrovascular disease as complications among patients with CKD (4); complications with a risk of acute kidney injury and cardiovascular-kidney comorbidity  are recent pathological features (6).Recent large-scale analysis of a Japanese real-world hospital database has shown that the presence of CKD or HF, in addition to type 2 diabetes, significantly increased mortality (9). It is issued by the Ministry, encouraging the conduction of awareness-raising activities regarding the importance of early detection and treatment of CKD (9).Therefore, the report of the study group on Measures against Kidney Disease, which was created in close cooperation with academic societies such as the Japanese Society of Nephrology, Japanese Circulation Society, Japan Diabetes Society, Japan Medical Association, local governments, and patient groups, was published by Health, Labor, and Welfare Association in July 2018 (2). Subsequently, the aging of patients with chronic dialysis has been noticeable in recent years, and the statistical analysis survey results by the Japanese Society for Dialysis Medicine [the annual report of The Japanese Society for Dialysis Therapy (JSDT) Renal Data Registry (JRDR)] also revealed that the number of patients with chronic dialysis would reach approximately 350,000 by the end of 2021, with an increasing trend in patients aged ≥70 years (11). Particularly, patients undergoing chronic dialysis are reported to have high mortality and hospitalization rates. Functional dependence is clinically recognized as a contributing factor to subsequent disability, recurrent hospitalization, and increased mortality (12). An increasing burden of functional dependence has been observed in several studies with progressive deterioration of kidney function; functional dependence may contribute to morbidity and mortality in patients with chronic dialysis, suggesting a problem from the socio-medical viewpoint in Japan and other countries (13).Moreover, the number of Japanese patients with CKD is estimated to be approximately 13.3 million, of which approximately 1 in 8 adults has CKD, and the prevalence of CKD is high among the elderly (2). Kidney function is assessed using glomerular filtration rate (GFR) and is calculated using the Japanese GFR estimation formula (eGFR-Cr) from serum creatinine level, gender, and age in daily clinical practice(14); additionally, the Japanese GFR estimation formula (eGFR-cysC) based on serum cystatin C levels can be used (15). If more accurate renal function is required, measured GFR is used using inulin clearance. Creatinine is produced by muscle, and serum creatinine levels are affected by muscle mass, so eGFR-Cr is estimated to be high in patients with decreased muscle mass due to sarcopenia  in elderly people, muscle disease, or limb loss (2). Conversely, eGFR-Cr is estimated to be low in cases such as athletes who have a habit of exercising and a large amount of muscle mass (2). Serum creatinine levels are also affected by diet, exercise, and renal tubular secretion (2). Cystatin C is produced by cells throughout the body, and serum cystatin C levels are not affected by muscle mass but are influenced by thyroid function, smoking, inflammation, fat mass, pregnancy, and immunosuppressants．Although CKD often presents a few subjective symptoms, it can be easily diagnosed by blood and urine tests. Therefore, the prevention of severity and suppression of CVD onset through early diagnosis, via health checkups, tests at medical institutions, and providing appropriate treatment, is important (16). Uemura et al described the accuracy of eGFR-cysC, when compared with eGFR-Cr, in patients with SMID. In their study, the authors measured 24h-Ccr in 18 patients with SMID and converted it into inulin clearance (Cin), which is the gold standard for measuring GFR, by an equation developed from children (16). The authors reported that eGFR-Cr does not reliably estimate kidney function in adult patients with SMID and alternatively recommended using cystatin C (cysC)-based eGFR (eGFR-cysC) in the Japanese population of severe motor and intellectual disabilities (16). This study was well performed and the results were important and interesting, especially for practitioners and researchers in the fields of kidney medicine and gerontology (16).Calculating eGFR in patients with severe motor and intellectual disabilities (SMID) using serum creatinine overestimates kidney function due to low muscle mass. Thus, in this issue of JMA Journal, Uemura O et al aimed to evaluate and compare the efficacy of eGFR formulae in patients with SMID by evaluating the efficacy of three eGFR equations, creatinine clearance (Ccr)-based eGFR (eGFR-Cr), serum cystacin C (cysC)-based eGFR (eGFR-cysC), and serum beta-2 microglobulin (β2MG)-based eGFR (eGFR-β2MG), in 18 patients with SMID NoneKouichi Tamura is one of the Editors of JMA Journal and on the journal’s Editorial Staff. He was not involved in the editorial evaluation or decision to accept this article for publication at all.


Given that the JAK pathway constitutes a pivotal regulator of pro-inflammatory signal transduction, JAK inhibitors are considered to ameliorate the effects of several cytokines and provide a broader level of control in systemic auto-immune diseases beyond inflammatory arthritides.4 and non-infectious scleritis5 are presented. Takayasu arteritis is chronic systemic inflammatory condition characterized by vascular granulomatous inflammation leading to progressive intimal fibrosis, vessel-wall remodelling, and eventually stenosis and aneurysm formation in large vessels. Takayasu arteritis is associated with severe (cardio)vascular morbidity and mortality due to suboptimal control of vascular inflammation, the relapsing pattern of the disease and the adverse consequences of high cumulative steroid doses, which currently represent the cornerstone of treatment.6 More importantly, the lack of sufficient evidence in randomised control trials supporting the administration of conventional and biologic disease modifying drugs which are commonly used in daily practice highlights the unmet therapeutic needs in this condition.7In the current issue of the Mediterranean Journal of Rheumatology, two case series of successful treatment with tofacitinib - a non-selective JAK1/2/3 inhibitor – administered “off-label” in refractory cases of Takayasu arteritis8 Taking into account that the synthesis of each cytokine is driven by the JAK pathway, it is not surprising that tofacitinib reduced cytokine production and inhibited both T-cell and macrophage activation in a mouse model of large vessel vasculitis.9 Such data indicate that tofacitinib may be useful for the treatment of Takayasu disease, particularly in cases unresponsive to conventional treatment. Prakashini MV et al.4 report 8 cases of refractory Takayasu arteritis effectively controlled after the administration of tofacitinib 5mg bd. This is in line with other observations in the whole spectrum of large vessels vasculitis.10 suggesting that multicytokine blockade through JAK inhibition may be a promising option for the effective suppression of vascular inflammation in these conditions.11A plethora of innate and adaptive immune cells contribute to the pathogenic process of vascular remodelling in Takayasu arteritis. For example, enriched interferon-α and interferon-γ signatures have been reported in both residual CD4 and CD8 T-cells whilst interleukins −6 and −12 are aberrantly expressed by macrophages and monocytes in vascular lesions of patients suffering from the disease.5 present three treatment-resistant cases of bilateral idiopathic scleritis in which remission was achieved after treatment with tofacitinib.Non-infectious inflammatory scleritis is an inflammatory ocular disease occurring as idiopathic or in the context of systemic inflammatory diseases. Although biologic drugs, namely, adalimumab, infliximab, and rituximab have been validated as targeted therapies in individuals suffering from systemic disorders, relapse may occur and lead to vision-threatening ocular complications. Dey at al.12 In this regard, tofacitinib may exert anti-inflammatory properties in both the ocular surface and intraocular inflammation by blocking the development and differentiation of TH1 cells and reducing the production of interferon-1 as demonstrated in animal models of experimental autoimmune uveitis.13 Similar observations have been reported with upadacitinib, a selective JAK-1 Inhibitor.14 Such experimental findings have been confirmed in a number of cases and short series describing the beneficial effects of JAK inhibition in patients suffering from a broad spectrum of inflammatory eye disorders.15The pathophysiology of non-infectious scleritis remains largely unknown but immune activation of different cells populations including macrophages, Th1 and Th17 T-cells subsets as well as B- cells are pivotal drivers of the residual inflammatory process in sclera tissue.17 However, the complexity of immune-mediated diseases such as Takayasu arteritis and inflammatory scleritis in which numerous cytokines and various cell types interact with each other suggest that inhibition of multiple cytokine signalling pathways with tofacitinib rather that single cytokine blockade may be clinically valuable. Data from randomised trials and prospective studies will provide further evidence for the efficacy and safety of JAK inhibitors as their use is expanding in a diverse range of indications.Despite the expanding use of JAK inhibitors across different medical specialties several safety concerns have been raised regarding herpes zoster infections, cardiovascular, and thromboembolic side effects as well as cancer occurrence.


The endocannabinoid system (ECS) plays a key neuromodulatory role in the brain. Main features of endocannabinoids (eCBs) are that they are produced on demand, in response to enhanced neuronal activity, act as retrograde messengers, and participate in the induction of brain plasticity processes. Sexual activity is a motivated behavior and therefore, the mesolimbic dopaminergic system (MSL) plays a central role in the control of its appetitive component (drive to engage in copulation). In turn, copulation activates mesolimbic dopamine neurons and repeated copulation produces the continuous activation of the MSL system. Sustained sexual activity leads to the achievement of sexual satiety, which main outcome is the transient transformation of sexually active male rats into sexually inhibited animals. Thus, 24 h after copulation to satiety, the sexually satiated males exhibit a decreased sexual motivation and do not respond to the presence of a sexually receptive female with sexual activity. Interestingly, blockade of cannabinoid receptor 1 (CB1R) during the copulation to satiety process, interferes with both the appearance of the long-lasting sexual inhibition and the decrease in sexual motivation in the sexually satiated males. This effect is reproduced when blocking CB1R at the ventral tegmental area evidencing the involvement of MSL eCBs in the induction of this sexual inhibitory state. Here we review the available evidence regarding the effects of cannabinoids, including exogenously administered eCBs, on male rodent sexual behavior of both sexually competent animals and rat sub populations spontaneously showing copulatory deficits, considered useful to model some human male sexual dysfunctions. We also include the effects of cannabis preparations on human male sexual activity. Finally, we review the role played by the ECS in the control of male sexual behavior expression with the aid of the sexual satiety phenomenon. Sexual satiety appears as a suitable model for the study of the relationship between eCB signaling, MSL synaptic plasticity and the modulation of male sexual motivation under physiological conditions that might be useful for the understanding of MSL functioning, eCB-mediated plasticity and their relationship with motivational processes. The World Health Organization (WHO) recognizes that “sexuality is a central aspect of being human and encompasses sex, gender identities and roles, sexual orientation, eroticism, pleasure, intimacy and reproduction” and states, with robust research evidence, that sexual health positively correlates with mental health and life quality . In humaSince some of the brain circuits involved in different aspects of sexual behavior expression are highly conserved through evolution, the understanding of the central regulation of male sexual behavior derives substantially from preclinical studies mostly obtained in rodents. The use of animal models of human behavior reduces the practical and ethical issues that human studies represent. Also, establishing the role played by the different neurotransmitter systems, as well as the participation of specific brain areas in the control of male sexual activity, and other neurobiological bases of sexual behavior can only be studied through experimental models. Rodent male sexual behavior is considered to show good face validity, i.e., the consideration that the model is analogous or homologous to human behavior .There are several neurotransmitters involved in the regulation of male rat sexual behavior and the actions of these neurotransmitters are highly regulated by other chemical messengers exerting modulatory effects. The endocannabinoid system (ECS) is one important neuromodulator of brain neurotransmitter actions. Here, we will discuss ongoing work investigating how endocannabinoid (eCB) signaling influences male sexual behavior (MSB) expression by modulating the activity of the mesolimbic dopaminergic system (MSL) and will review the effects of exogenously administered eCBs on the sexual behavior display of different male rat sexual sub populations, spontaneously showing copulatory deficits. Specifically, we will describe the sexual behavior of male rats and its the central regulation. We also summarize some of the models used for the study of different aspects of male sexual activity and the distinct sexual sub populations we can find in rats . In the next section, we provide an overview of the ECS (Section “3. The endocannabinoid system”) and thereafter we review the available knowledge on cannabinoid effects on male sexual behavior, including the effects of Cannabis preparations on male human sexuality. We also review the involvement of the ECS in the control of MSB expression in sexually proficient animals . Thereafter, we summarize the available data on a possible role of the ECS in the expression of MSB in sexually experienced rats (Section “5. Role of the ECS in the regulation of MSB expression”). We then expand the knowledge on this role of eCBs to a model of sexual inhibition: sexual satiety . To that aim we first describe the sexual satiety phenomenon and its relationship with the MSL system. We continue reviewing current knowledge on the role played by the ECS at the MSL circuit in the induction of brain plastic changes associated to the induction of this phenomenon. We conclude that sexual satiety is a model that allows the study of MSL system functioning, eCB-mediated plasticity, and its relationship with motivational processes under physiological conditions (Section “7. Concluding remarks”).Sexual behavior in male rats consists in the presentation of various sexually related anticipatory behaviors that include female anogenital investigation, sniffing and licking, as well as the emission of 50 kHz ultrasonic vocalizations. These behaviors are followed by the display of a highly stereotyped copulatory pattern. Male rat copulatory pattern is shaped by three distinct motor behaviors : (i) mouDuring sexual performance, the male rat presents several mounts and intromissions before achieving ejaculation. After ejaculating, male rats exhibit a transient period of sexual refractoriness, named the post-ejaculatory interval, which ends when they are able to renew sexual intercourse, indicated by the presentation of an intromission that initiates a new copulatory series. The copulatory responses displayed from the start of the first intromission until ejaculation constitute an ejaculatory series and, when the post-ejaculatory interval is included, it is named copulatory series .Although innate, the copulatory pattern becomes more efficient and stable through sexual experience, in such a way that its temporal development  and the stimulation needed to achieve ejaculation (number of pre-ejaculatory mounts and intromissions) become very similar among subjects, allowing the definition of efficient sexual activity through the establishment of baseline sexual measures. These stable basal copulatory measures serve as control parameters for comparisons with the effects of experimental manipulations on male rat sexual behavior. Thus, sexually experienced male rats, defined as those achieving ejaculation in less than 15 min, in at least three of five independent sexual training sessions, are used as control group. The most common sexual parameters used to evaluate male rat copulatory behavior are described in Studies on male rat sexual activity have made a distinction between its motivational and consummatory components . Sexual Male rodents share with men the capacity to engage in sexual activity continuously during their adult life. In addition, the brain structures, neural networks, and neurochemical control of male sexual activity appear to be common to both species . BesidesMale rat sexual behavior can be analyzed in the laboratory with different approximations, and a variety of models have been proposed for the study of specific aspects of male sexual activity including sexual arousal, sexual motivation and performance, and sexual inhibition. Some of the models addressing specific aspects of sexual activity are briefly described.Erection is considered an indicator of sexual arousal in male rats and men, and the mechanisms involved in this sexual response are importantly shared between both species . The nonSexual incentive motivation, considered to model human sexual desire , is trigThe most common analysis to assess male rat sexual performance consists of the recording of sexual activity in sexually experienced male rats during a single copulatory series, however, it has been proposed that a more extended observation period (30 min), allowing the display of two to three successive copulatory series, offers a better panorama . SexuallInhibition of sexual behavior display in sexually competent male rats can be achieved through aversive conditioning , howeverMale rat’s natural biological variability permitted the identification of sexual sub populations, which could serve as preclinical models to study some human sexual dysfunctions. These sub populations include sexually inactive males (non-copulators), sexually sluggish males, and rapid ejaculators .Sexually inactive male rats are sexually mature, healthy, apparently normal animals that do not mate when sexually receptive females are accessible, in spite of being repeatedly exposed to them. These animals are referred to as non-copulators (NC); they have intact genital responses (erection and ejaculation)  and normSexually sluggish male rats and rapid ejaculators have been considered to represent the two extremes of the Gaussian distribution of ejaculatory endophenotypes , proposeAll central nervous system functions, from reflexes to complex behaviors, ultimately result from the capability of neurons to electrochemically communicate with each other. Communication between neurons occurs at tiny gaps called synapses, where specialized parts of the two cells  come very close (a few nanometers) of one another, to allow for chemical transmission . A wide The ECS has emerged as an important and widespread neuromodulatory system capable of regulating the levels and activity of most of the other neurotransmitter systems, when it is needed, providing immediate feedback. The ECS comprises cell membrane receptors (cannabinoid receptors) and their endogenous ligands (endocannabinoids) along with the enzymes and molecules required for their synthesis and degradation .Cnr1 gene, and the protein consists of 472 amino acids, while the CB2R is encoded by the Cnr2 gene, and the protein consists of 360 amino acids in humans. At the protein level, CB1R and CB2R have 44% sequence homology (The ECS includes two receptor subtypes known as cannabinoid receptor 1 (CB1R) and cannabinoid receptor 2 (CB2R). The CB1R is encoded by the homology . Both CBhomology . In addihomology .At the central nervous system (CNS) CB1R are especially abundant in the cortex, basal ganglia, hippocampus, and cerebellum . The gre9-tetrahydrocannabinol (Δ9-THC), hence its name. Both, eCBs and phytocannabinoids (molecules derived from Cannabis), bind to and activate cannabinoid receptors (CBR) , are lipid signaling molecules, which have a structural similarity to molecules derived from the Cannabis plant, such as Δrs (CBR) . N-arachrs (CBR) .The intrinsic efficacy of these eCBs for CBR also varies. 2-AG is a full agonist with moderate-to-low affinity for both CBR, whereas AEA is a high-affinity partial agonist of CB1R, and a very-low-efficacy agonist of the CB2R . Under cBoth AEA and 2-AG are synthetized “on demand” and released immediately, without storage in vesicles . RetrogrThe literature related to the effects of phytocannabinoids and exogenously administered eCBs on male sexual behavior (MSB) is scarce. In these studies, some data are more indirect than others, and in some cases, MSB analysis was not the main purpose of the referenced study and therefore the findings regarding MSB were not discussed in depth. In the next section we summarize and integrate the information available about this topic, considering that all data contribute to a certain extent to the understanding of the effects of targeting this neuromodulatory system on a complex behavior like MSB.9-THC, since this molecule is recognized as the most important psychoactive compound of Cannabis preparations. We review here data on the sexual effects of CBR agonists in sexually active male rodents, which are summarized in 9-THC; a third set summarizes the sexual effects of synthetic cannabinoids, and at the end those produced by exogenously administered eCBs  in male mice and, as a consequence, no sexual behavior was observed. In this case and taking into account that the temporal sexual parameters were the most affected by CB1R agonists, it is possible that the observation period (10 min) was not sufficient to detect changes in sexual behavior. This is a probable explanation since in another set of experiments performed by the same research group, it was reported that even though during the observation period (20 min) mounting behavior was not observed, the next day both vehicle- and drug-treated females showed seminal plugs, evidencing that the males eventually showed sexual activity , which they considered indicative of sexual motivation. More interesting, by the third day of treatment, in addition to tolerance to the sedative effects, the number of mice showing “attempted mount” was significantly larger than in the vehicle-treated group, suggesting tolerance development to Δ9-THC sexual effects. Also, the number of animals showing mounts and intromissions was slightly increased in Δ9-THC-treated males in comparison with control mice  than during the last evaluation (day 14) . Another9-THC  decreased sexually motivated behavior and social investigation (9-THC (3X/week) for 3 weeks significantly increased ML and IL, however, after 7 weeks of treatment ML was the only parameter still augmented  along 60 days, either did not assess MSB specific parameters or did not find differences with the control group , suggesting that AEA acute administration was able to transform the animals’ sexual condition from inactive into sexually active . In sexuThe most direct strategy to understand the role played by the ECS in MSB expression is its manipulation under physiological conditions with the aim of pharmacological tools. One approach is to interfere with eCB’s putative actions during sexual activity by blocking CBR with specific antagonists; if this manipulation modifies a specific sexual response, a physiological role for eCBs in that behavior can be inferred. Another approach is to increase the eCB tone by inhibiting the reuptake and/or degradation of specific eCBs .The available data show that antagonism of CB1R with low AM251 doses lacked sexual effects , similarInfusion of the CB1R antagonist SR 141716A (Rimonabant) into the paraventricular hypothalamic nucleus (PVN) has been found to induce spontaneous penile erection, an effect apparently mediated by the activation of oxytocinergic transmission, which is considered to indicate sexual arousal. However, infusion of CB1R agonists into the PVN lacked effects on this sexual reflex , 2006.Together, data suggest that the ECS plays no predominant role in the modulation of MSB of sexually proficient animals but rather becomes relevant when its functioning is altered, as in NC and sexually sluggish animals, which condition most probably involves neurotransmitter imbalances. This puts forward the potentiality of targeting the ECS in the search for new therapeutic agents for the treatment of sexual dysfunctions, as suggested for AEA in the management of lifelong delayed ejaculation .2+ concentrations that might result from this augmented neuronal activity or from the direct activation of metabotropic receptors coupled to phospholipase C, like group I metabotropic glutamate receptors (mGluR1 and mGluR5)  . It is c mGluR5) . In the Sexually experienced male rats allowed to copulate without restriction with a single sexually receptive female will ejaculate repeatedly (seven successive ejaculations in average) until becoming sexually satiated . Sexual This sexual inhibitory state remains well established for 48 h after copulation to satiety and is expressed in the majority of the males by the absence of sexual activity in the presence of a sexually receptive female . The absTwenty-four hours after copulation to satiety, the well-established sexual inhibitory state can be pharmacologically reversed by different drug treatments see  that havThe MSL circuit is essential for the processing of natural rewarding behaviors , like seThe activity of the dopaminergic neurons of the MSL system is regulated at the VTA, where the ECS components i.e., receptors and ligands as well as eCB synthetizing and degrading enzymes, are expressed .There is sound evidence for the activation of the MSL system by male rat sexual activity, indicated by the increase in NAcc dopamine extracellular concentrations , augmentMicrodialysis studies showed that MSL dopamine transmission is altered 24 h after copulation to satiety, since NAcc dopamine basal concentrations were significantly diminished in sexually satiated males when compared to non-satiated rats. Moreover, exposure of the sexually satiated males at that time point to a receptive female did not induce the typical increase in NAcc dopamine release but rather dopamine concentrations decreased further . This blThe activity of the dopaminergic neurons of the MSL system is regulated at the VTA, a brain region mainly composed of dopamine neurons (55−65%) and GABA interneurons (30%) but also containing a small proportion of glutamatergic neurons (5%) . In thisDopamine release at the NAcc depends both on dopamine neuron activity  and the The main mechanisms reducing or inhibiting NAcc dopamine terminal release involve: (i) The D2 autoreceptor-mediated feedback  inhibitiAn additional level of control of mesolimbic dopamine neuron activity is exerted by the eCB release in the VTA that emerges during prolonged or heightened dopamine neuron discharge . The relEndocannabinoid-mediated regulation of MSL dopamine neuron activity appears particularly relevant to the sexual satiety-associated changes in MSL system functioning, as these lipid messengers play a role in the induction of the long-lasting physiological and behavioral changes of the sexually satiated rats .Investigating the possible mechanisms mediating these eCB actions have been the matter of our latest interest. Based on the evidence showing that eCBs induce long-term synaptic plasticity phenomena at VTA GABAergic  and glutThe VTA neurons that are activated during copulation, identified by c-Fos protein expression, receive glutamatergic inputs . Thus, sGlutamatergic plasticity involves changes in the density and subunit composition of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors . We rece2+, while GluA2-lacking AMPARs are Ca2+- permeable. The repeated activation of GluA2-lacking AMPAR triggers a long-lasting switch in their composition to GluA2-containing AMPARs  to follow the transient, but long-lasting, change in the functioning of the MSL system triggered by its repeated activation by a natural stimulus under physiological conditions; (ii) to identify the mechanisms involved in changing MSL dopamine neuron function, and (iii) the impact of such changes on motivational states. Moreover, the reversible nature of the long-lasting physiological changes that accompany the sexual satiety phenomenon offers the possibility to detect unidentified mechanisms capable of restoring MSL system functioning.We can conclude from the reviewed data. On the one side, that exogenously administered eCBs modulate MSB expression. Although AEA and 2-AG exert their sexual effects through the activation of a same receptor, the CB1R, their pharmacological profile differs. Thus, AEA but not 2-AG effects on MSB are biphasic, with low doses facilitating and high doses inhibiting or losing their facilitative effects on copulation. The modulatory actions of eCBs on MSB become more evident in males with sexual deficiencies, in which exogenously administered eCBs restore those deficiencies either transiently or in a long-lasting manner. On the other side, eCBs play a physiological role in the control of MSB expression in animals that copulate to satiety, through the induction of plastic changes at the MSL system that could represent a protective mechanism against the excessive activation produced by heightened natural rewarding stimulation. The sexual satiety phenomenon appears as a natural model for the study of MSL system functioning and its relationship with motivational processes. The knowledge obtained from the analysis of the sexual satiety phenomenon might shed light on different aspects of the neurobiology of MSB in men, as well as on the MSL circuit mechanisms underlying human motivational functions and dysfunctions associated with neuropsychiatric disorders.Both authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.