Supporting parenting among Syrian refugees in Lebanon: a randomized controlled trial of the caregiver support intervention.

BACKGROUND: Parenting interventions in humanitarian settings have prioritized the acquisition of parenting knowledge and skills, while overlooking the adverse effects of stress and distress on parenting-a key mediator of refugee children's mental health. We evaluated the effectiveness of the Caregiver Support Intervention (CSI), which emphasizes caregiver wellbeing together with training in positive parenting. METHODS: We conducted a two-arm randomized controlled trial of the CSI with Syrian refugees in Lebanon, with an intent-to-treat design, from September 2019-December 2020. A total of 480 caregivers from 240 families were randomized to the CSI or a waitlist control group (1:1). Retention from baseline to endline was 93%. Data on parenting and caregiver psychological wellbeing were collected at baseline, endline, and three-month follow-up. Prospective trial registration: ISRCTN22321773. RESULTS: We did not find a significant change on overall parenting skills at endline (primary outcome endpoint) (d = .11, p = .126) or at follow-up (Cohen's d = .15, p = .054). We did find a significant effect on overall parenting skills among participants receiving the full intervention-the sub-sample not interrupted by (COVID-19) (d = 0.25, p < .05). The CSI showed beneficial effects in the full sample at endline and follow-up on harsh parenting (d = -.17, p < .05; d = .19, p < .05), parenting knowledge (d = .63, p < .001; d = .50, p < .001), and caregiver distress (d = -.33, p < .001; d = .23, p < .01). We found no effects on parental warmth and responsiveness, psychosocial wellbeing, stress, or stress management. Changes in caregiver wellbeing partially mediated the impact of the CSI on harsh parenting, accounting for 37% of the reduction in harsh parenting. CONCLUSIONS: The CSI reduced harsh parenting and caregiver distress, and demonstrated the value of addressing caregiver wellbeing as a pathway to strengthening parenting in adversity. These effects were achieved despite a pandemic-related lockdown that impacted implementation, a severe economic crisis, and widespread social unrest. Replication under less extreme conditions may more accurately demonstrate the intervention's full potential.

Open-source method of image cytometry in dorsal root ganglia tissue with immunofluorescence.

Immunohistochemistry (IHC) is a valuable tool in clinical and biological research for evaluating proteins and other antigens in spatially bound tissue. In neuroinflammatory pain research, primary afferent neurons of the dorsal root ganglion (DRG) are studied to understand molecular signaling mechanisms involved in nociception (pain) and inflammation. Measuring IHC (immunofluorescence) in DRG neurons requires manual hand tracing of nuclear and somatic boundaries, which is laborious, error-prone, and may require several weeks to collect the appropriate sample size with a mouse or pen-input display monitor. To overcome these limitations and increase standardization of sampling and measurement, we employed a reliable neuronal cytoplasmic reporter, exclusive to DRG neuronal soma, in a semi-automated algorithm-based approach of Image Cytometry in rat DRG (IC-DRG). The resulting output images are binary nuclear and somatic masks of DRG neurons, defining boundaries of measurement for CellProfiler and manually scored at 94% accurate. Herein, we successfully show a novel approach of automated image analysis for DRG neurons using a robust ImageJ/FIJI script, overcoming morphological variability and imaging artifacts native to imaging frozen tissue sections processed with immunofluorescence.

Problems after flight: understanding and comparing Syrians' perspectives in the Middle East and Europe.

BACKGROUND: Syrian refugees and asylum seekers (SRAs) face multiple stressors after flight, which may vary due to different geographic, economic, cultural and socio-political contexts in the host countries. Past research has recognised the importance of participants' own perspectives. The aims of this multi-country study were to identify and compare self-reported problems of SRAs between various settings. METHODS: A semi-structured client-generated outcome measurement was used to collect data among adult SRAs in Jordan (N = 61), Turkey (N = 46) and Switzerland (N = 57) between September 2018 and November 2019. Answers were analysed following thematic analysis. RESULTS: Over half of the participants reported practical problems with an emphasis on camp-related problems (Jordan), finances (Turkey), employment (Jordan and Switzerland) and government regulations (Switzerland), followed by psychological, and social issues. CONCLUSION: This study highlights the impact of local contextual factors on wellbeing. The findings emphasise that planning preventative procedures and mental health care services for SRAs need to consider local challenges affecting the population in specific countries.

Determinants of Children's Mental Health in War-Torn Settings: Translating Research Into Action.

Research on the mental health and psychosocial wellbeing of children in conflict-affected settings has undergone a significant paradigm shift in recent years. Earlier studies based on a war exposure model primarily emphasized the effects of direct exposure to armed conflict; this has gradually given way to a broader understanding of the diverse pathways by which organized violence affects children. A robustly supported comprehensive model includes risk factors at multiple points in time (prior war exposure, ongoing daily stressors) and at all levels of the social ecology. In particular, findings suggest that material deprivation and a set of family variables, including harsh parenting, parental distress, and witnessing intimate partner violence, are important mediators of the relationship between armed conflict and children's wellbeing. To date, however, interventions aimed at supporting war-affected children's wellbeing, both preventive and treatment-focused, have focused primarily on direct work with children, while paying only modest attention to ongoing risk factors in their families and broader environments. Possible reasons for the ongoing prioritization of child-focused interventions are considered, and examples are provided of recent evidence-based interventions that have reduced toxic stressors (harsh parenting and the use of violent discipline by teachers) in conflict-affected communities.

Strategies to improve the quality and usefulness of mental health trials in humanitarian settings.

A striking rise in the number of people affected by humanitarian crises has led to an increase in mental health and psychosocial support interventions to reduce the psychological effects of such crises. In a parallel trend, researchers have brought increased methodological rigour to their evaluation of these interventions. However, several methodological issues still constrain the quality and real-world relevance of the existing evidence base. We examine five core challenges in randomised controlled trials of mental health and psychosocial support interventions with conflict-affected and disaster-affected populations. These challenges are: translating intervention effects into metrics of real-world significance; giving adequate consideration to the selection and monitoring of control conditions; following rigorous processes to ensure outcome measures are culturally appropriate and psychometrically sound; ensuring and monitoring implementation variables, including fidelity, exposure, participant engagement, and the competence of implementation staff; and assessing mechanisms of change.

Afghan mental health and psychosocial well-being: thematic review of four decades of research and interventions.

BACKGROUND: Four decades of war, political upheaval, economic deprivation and forced displacement have profoundly affected both in-country and refugee Afghan populations. AIMS: We reviewed literature on mental health and psychosocial well-being, to assess the current evidence and describe mental healthcare systems, including government programmes and community-based interventions. METHOD: In 2022, we conducted a systematic search in Google Scholar, PTSDpubs, PubMed and PsycINFO, and a hand search of grey literature (N = 214 papers). We identified the main factors driving the epidemiology of mental health problems, culturally salient understandings of psychological distress, coping strategies and help-seeking behaviours, and interventions for mental health and psychosocial support. RESULTS: Mental health problems and psychological distress show higher risks for women, ethnic minorities, people with disabilities and youth. Issues of suicidality and drug use are emerging problems that are understudied. Afghans use specific vocabulary to convey psychological distress, drawing on culturally relevant concepts of body-mind relationships. Coping strategies are largely embedded in one's faith and family. Over the past two decades, concerted efforts were made to integrate mental health into the nation's healthcare system, train cadres of psychosocial counsellors, and develop community-based psychosocial initiatives with the help of non-governmental organisations. A small but growing body of research is emerging around psychological interventions adapted to Afghan contexts and culture. CONCLUSIONS: We make four recommendations to promote health equity and sustainable systems of care. Interventions must build cultural relevance, invest in community-based psychosocial support and evidence-based psychological interventions, maintain core mental health services at logical points of access and foster integrated systems of care.

Subcellular localization of neuronal nuclei (NeuN) antigen in size and calcitonin gene-related peptide (CGRP) populations of dorsal root ganglion (DRG) neurons during acute peripheral inflammation.

Pseudo-unipolar cell bodies of somatosensory primary neurons are located in the dorsal root ganglia (DRG). The somatic and peripheral domains of DRG neurons are often studied in sensory pain research to understand molecular mechanisms involved in the activation of pain and maintenance of inflammation. Adjuvant-induced arthritis (AIA) is an inflammatory model that elicits a robust and rapid onset immune response with a maximal swelling period of 24-48 h and persisting for several weeks. The AIA model in the hind paw of the rat elicits a potent inflammatory response of the dermis and epidermis, leading to protein expression changes for sensitization of many DRG neurons; however, it is unknown if the AIA model in the hind paw of the rat induces DRG neuronal injury, necrosis, or apoptosis at the somatic level. Neuronal nuclei (NeuN) antigen is a biomarker for post-mitotic neurons, neuronal identification, protein alterations, injury, and loss. Calcitonin gene-related peptide (CGRP) is expressed in C and Aδ DRG neurons, a subset of DRG neurons known to play a role in peripheral sensitization. The focus of this research was to evaluate the expression pattern of NeuN immunoreactivity, in size (soma) and CGRP subpopulations of DRG neurons in naïve and inflamed groups. Confirmed by both immunofluorescence and immunoprecipitation, DRG neuronal expression of NeuN was localized to nuclear and cytoplasmic subcellular compartments. NeuN increased within the nucleus of small CGRP positive DRG neurons during inflammation, indicating a potential role for NeuN in a subset of nociceptive neurons.

Separation of Rat Epidermis and Dermis with Thermolysin to Detect Site-Specific Inflammatory mRNA and Protein.

Easy-to-use and inexpensive techniques are needed to determine the site-specific production of inflammatory mediators and neurotrophins during skin injury, inflammation, and/or sensitization. The goal of this study is to describe an epidermal-dermal separation protocol using thermolysin, a proteinase that is active at 4 °C. To illustrate this procedure, Sprague Dawley rats are anesthetized, and right hind paws are injected with carrageenan. Six and twelve hours after injection, rats with inflammation and naïve rats are euthanized, and a piece of hind paw, glabrous skin is placed in cold Dulbecco's Modified Eagle Medium. The epidermis is then separated at the basement membrane from the dermis by thermolysin in PBS with calcium chloride. Next, the dermis is secured by microdissection forceps, and the epidermis is gently teased away. Toluidine blue staining of tissue sections show that the epidermis is separated cleanly from the dermis at the basement membrane. All keratinocyte cell layers remain intact, and the epidermal rete ridges along with indentations from dermal papillae are clearly observed. Qualitative and real-time RT-PCR is used to determine nerve growth factor and interleukin-6 expression levels. Western blotting and immunohistochemistry are finally performed to detect amounts of nerve growth factor. This report illustrates that cold thermolysin digestion is an effective method to separate epidermis from dermis for evaluation of mRNA and protein alterations during inflammation.

Growing pains: the impact of disaster-related and daily stressors on the psychological and psychosocial functioning of youth in Sri Lanka.

Daily stressors may mediate the relation between exposure to disaster-related stressors and psychological and psychosocial distress among youth in disaster-affected countries. A sample of 427 Sri Lankan Sinhalese, Tamil, and Muslim youth (mean age = 14.5) completed a survey with measures of exposure to disaster-related stressors and daily stressors, psychological distress (posttraumatic stress, depression, and anxiety), and psychosocial distress. The results indicated that daily stressors significantly mediated relations between war- and tsunami-related stressors and psychological and psychosocial distress. Some daily stressors not directly related to disaster also predicted functioning. These results point to the need for policies and interventions that focus on reducing proximal daily stressors that are salient to Sri Lankan youth exposed to disasters.

Protocol for a randomized control trial of the caregiver support intervention with Syrian refugees in Lebanon.

BACKGROUND: There is evidence that chronic stress negatively impacts parenting among refugees and other war-affected communities. Persistent parental stress and distress may lead to unresponsive, anxious, or overly harsh parenting and a corresponding increase in emotional and behavior problems among children. Most parenting interventions emphasize the acquisition of knowledge and skills; however, this overlooks the deleterious effects of chronic stress on parenting. The Caregiver Support Intervention (CSI) aims to strengthen quality of parenting skills by lowering stress and improving psychosocial wellbeing among refugee caregivers of children aged 3-12 years, while also increasing knowledge and skills related to positive parenting. The CSI is a nine-session psychosocial group intervention delivered by non-specialist providers. It is intended for all adult primary caregivers of children in high-adversity communities, rather than specifically targeting caregivers already showing signs of elevated distress. METHODS/DESIGN: The primary objective of this study is to assess the effectiveness of the CSI through a parallel group randomized controlled study with Syrian refugee families in North Lebanon. Participants will be primary caregivers of children aged 3-12 years, with one index child per family. Families will be randomized to the CSI or a waitlist control group. A total of 240 families (480 caregivers) will be recruited into the study. Randomization will be at the family level, and CSI groups will be held separately for women and men. The study will be implemented in two waves. Outcomes for both arms will be assessed at baseline, post-intervention, and at a 3-month follow-up. The primary outcome is quality of parenting skills. Secondary outcomes include parental warmth and sensitivity, harsh parenting, parenting knowledge, and child psychosocial wellbeing. Putative mediators of the CSI on parenting are caregiver stress, distress, psychosocial wellbeing, and stress management. DISCUSSION: This trial may establish the CSI as an effective intervention for strengthening parenting in families living in settings of high adversity, particularly refugee communities. TRIAL REGISTRATION: International Society for the Registration of Clinical Trials, ISRCTN22321773. Registered on 5 August 2019.

Supporting Syrian families displaced by armed conflict: A pilot randomized controlled trial of the Caregiver Support Intervention.

BACKGROUND: The impact of armed conflict and displacement on children's mental health is strongly mediated by compromised parenting stemming from persistently high caregiver stress. Parenting interventions for refugees emphasize the acquisition of parenting knowledge and skills, while overlooking the deleterious effects of chronic stress on parenting. War Child Holland's Caregiver Support Intervention (CSI) aims to strengthen parenting by lowering stress and improving psychosocial wellbeing among refugee parents, while also increasing knowledge and skill related to positive parenting. The CSI is a nine-session group intervention delivered by non-specialist providers. OBJECTIVE: We describe the findings of a two-arm pilot randomized controlled trial of the CSI with Syrian refugees in Lebanon. The primary aim was to test the feasibility of our study methodology prior to conducting a definitive RCT. METHODS: We recruited 78 families (151 parents), who were randomized to the CSI or a waitlist control group. Data were collected at baseline and post-intervention. RESULTS: Randomization was successful, retention was high (99 %), as was intervention completion (95 % among women, 86 % among men). Implementation fidelity was excellent. Blinding was largely, though not completely effective. The CSI group showed significantly increased parental warmth and responsiveness, decreased harsh parenting, lowered stress and distress, improved psychosocial wellbeing, and improved stress management. CSI parents reported increased child psychosocial wellbeing. Control families showed no significant change on any variable. CONCLUSIONS: Findings demonstrate the feasibility of our methodology for a definitive RCT, and suggest that the CSI shows promise as a scalable approach to strengthening parenting in refugee communities. Trial registration # ISRCTN33665023.

Strengthening parenting in conflict-affected communities: development of the Caregiver Support Intervention.

BACKGROUND: There is robust evidence that compromised parenting, stemming from persistently high stress, mediates the impact of war and displacement on children's mental health and psychosocial wellbeing. Parenting interventions generally prioritize the acquisition of parenting knowledge and skills, while under-attending to parental stress and distress. This paper describes the development of the Caregiver Support Intervention (CSI), a nine-session group intervention for conflict-affected parents of children aged 3-13, that aims to strengthen parenting both indirectly, by lowering stress and improving psychosocial wellbeing among parents, and directly, by increasing knowledge and skill related to positive parenting. METHODS: We describe the multi-phase, iterative process by which we developed the CSI, and illustrate the essential role of community input in shaping the intervention and strengthening its cultural fit and perceived usefulness. We used focus group data from participants in successive cycles of implementation, feedback, and revision, as well as quantitative data and expert consultation to develop a culturally and empirically grounded intervention. RESULTS: This mixed-method, iterative approach to intervention development enabled us to develop a psychosocial intervention for conflict-affected caregivers that is feasible, acceptable, and perceived by participants as useful in addressing their own wellbeing and their parenting. Focus group data support the underlying model in which caregiver wellbeing powerfully influences parenting. CONCLUSIONS: Programs aimed at strengthening parenting in conflict-affected communities should substantively address caregiver wellbeing. An iterative approach incorporating community feedback can help ensure intervention acceptability and feasibility. We also illustrate the feasibility of involving men in parenting interventions.

The validity and clinical utility of post-traumatic stress disorder in Afghanistan.

This study examined the validity and utility of PTSD among 320 adults in Afghanistan. Findings support the validity of PTSD in this cultural context: PTSD symptoms were highly prevalent, shared common variance, and correlated as expected with exposure to traumatic stress. However, only limited support was found for the clinical utility of PTSD. Other types of psychiatric symptomatology, including depression and a culturally specific measure of general distress, correlated more highly with traumatic stress than did PTSD; and PTSD accounted for limited variance in functioning beyond that explained by depression and general distress. Implications for research and intervention are considered.

Daily stressors, war experiences, and mental health in Afghanistan.

Working in Afghanistan's capital city of Kabul, the authors assessed the relative contribution of daily stressors and war-related experiences of violence and loss to levels of depression, PTSD, impaired functioning, and a culturally specific measure of general psychological distress. For women, daily stressors were a better predictor than war experiences of all mental health outcomes except for PTSD; for men, daily stressors were a better predictor of depression and functional impairment, while war experiences and daily stressors were similarly predictive of general distress. For men, daily stressors moderated the relationship between war experiences and PTSD, which was significant only under conditions of low daily stress. The study's implications for research and intervention in conflict and post-conflict settings are considered.

The Aerobic and Cognitive Exercise Study (ACES) for Community-Dwelling Older Adults With or At-Risk for Mild Cognitive Impairment (MCI): Neuropsychological, Neurobiological and Neuroimaging Outcomes of a Randomized Clinical Trial.

Prior research has found that cognitive benefits of physical exercise and brain health in older adults may be enhanced when mental exercise is interactive simultaneously, as in exergaming. It is unclear whether the cognitive benefit can be maximized by increasing the degree of mental challenge during exercise. This randomized clinical trial (RCT), the Aerobic and Cognitive Exercise Study (ACES) sought to replicate and extend prior findings of added cognitive benefit from exergaming to those with or at risk for mild cognitive impairment (MCI). ACES compares the effects of 6 months of an exer-tour (virtual reality bike rides) with the effects of a more effortful exer-score (pedaling through a videogame to score points). Fourteen community-dwelling older adults meeting screening criteria for MCI (sMCI) were adherent to their assigned exercise for 6 months. The primary outcome was executive function, while secondary outcomes included memory and everyday cognitive function. Exer-tour and exer-score yielded significant moderate effects on executive function (Stroop A/C; d's = 0.51 and 0.47); there was no significant interaction effect. However, after 3 months the exer-tour revealed a significant and moderate effect, while exer-score showed little impact, as did a game-only condition. Both exer-tour and exer-score conditions also resulted in significant improvements in verbal memory. Effects appear to generalize to self-reported everyday cognitive function. Pilot data, including salivary biomarkers and structural MRI, were gathered at baseline and 6 months; exercise dose was associated with increased BDNF as well as increased gray matter volume in the PFC and ACC. Improvement in memory was associated with an increase in the DLPFC. Improved executive function was associated with increased expression of exosomal miRNA-9. Interactive physical and cognitive exercise (both high and low mental challenge) yielded similarly significant cognitive benefit for adherent sMCI exercisers over 6 months. A larger RCT is needed to confirm these findings. Further innovation and clinical trial data are needed to develop accessible, yet engaging and effective interventions to combat cognitive decline for the growing MCI population. ClinicalTrials.gov ID: NCT02237560.

Beyond trauma-focused psychiatric epidemiology: bridging research and practice with war-affected populations.

This article examines the centrality of trauma-focused psychiatric epidemiology (TFPE) in research with war-affected populations. The authors question the utility of the dominant focus on posttraumatic stress disorder and other disorders of Western psychiatry, and they identify a set of critical research foci related to mental health work with communities affected by political violence. Core assumptions of TFPE and its roots in logical positivism and the biomedical model of contemporary psychiatry are explored. The authors suggest that an alternative framework--social constructivism--can serve as a bridge between researchers and practitioners by helping to refocus research efforts in ways that are conceptually and methodologically more attuned to the needs of war-affected communities and those working to address their mental health needs.

The Afghan symptom checklist: a culturally grounded approach to mental health assessment in a conflict zone.

This article describes a methodology for developing culturally grounded assessment measures in conflict and postconflict situations. A mixed-method design was used in Kabul, Afghanistan, to identify local indicators of distress and develop the 22-item Afghan Symptom Checklist (ASCL). The ASCL contains several indigenous items and items familiar to Western mental health professionals. The ASCL was pilot tested and subsequently administered to 324 adults in 8 districts of Kabul. It demonstrated excellent reliability (alpha=.93) and good construct validity, correlating strongly with a measure of exposure to war-related violence and loss (r=.70). Results of the survey indicate moderate levels of distress among Afghan men and markedly higher levels of distress and impaired functioning among women (and widows in particular).

Evaluating the Analgesic Effect of the GLS Inhibitor 6-Diazo-5-Oxo-L-Norleucine in Vivo.

Glutamate is an excitatory neurotransmitter, produced by its synthetic enzyme, glutaminase (GLS), and packaged by vesicular transporters (VGluT2) into synaptic vesicles. Primary sensory peripheral nerve and spinal synaptic terminals release glutamate during nociceptive (pain) signaling. In post-incisional and inflammation models in rats, GLS and VGluT2 production is elevated in dorsal root ganglion neuronal cell bodies and transported to peripheral and spinal terminals for increased glutamate synthesis and release. 6-Diazo-5-oxo-l-norleucine (DON) is a GLS inhibitor that produces long lasting pain relief when applied to the inflamed paw of arthritic rats, but its effect in a post-incisional model has not been evaluated. In this study, we examined the analgesic efficacy of DON in a surgical incision model by measuring thermal latency and mechanical allodynia. Following behavioral evaluation, we examined the skin for VGluT2, GLS and glutamate immunoreactivity (ir). Our findings revealed that VGluT2-ir is elevated in the stratum lucidum by approximately 19%, 64 hours post-surgical incision and attenuated by approximately 5.4% after the administration of DON. During that same period GLS-ir was elevated in dermal nerve fibers by 52% and was attenuated by approximately 27.9% after the application of DON. Additionally, glutamate-ir was elevated in epidermal nerve fibers by 35% after incision and attenuated by approximately 23% after the administration of DON. Behavioral testing 24 and 48 hours after a single local administration of DON showed five out of six animals having an analgesic response to mechanical allodynia, but not to thermal hyperalgesia. Following a surgical incision, the area of injury shows increased VGluT2-, GLS-, glutamate-ir, mechanical allodynia and no change in thermal latency. After the application of the GLS inhibitor, DON, nerve fiber of the skin showed decreased VGluT2, GLS, and glutamate-ir. Furthermore, post-incision DON treated animals exhibited decreased mechanical allodynia with no change in thermal latency when compared to control animals.

Characterization of glutamatergic neurons in the rat atrial intrinsic cardiac ganglia that project to the cardiac ventricular wall.

The intrinsic cardiac nervous system modulates cardiac function by acting as an integration site for regulating autonomic efferent cardiac output. This intrinsic system is proposed to be composed of a short cardio-cardiac feedback control loop within the cardiac innervation hierarchy. For example, electrophysiological studies have postulated the presence of sensory neurons in intrinsic cardiac ganglia (ICG) for regional cardiac control. There is still a knowledge gap, however, about the anatomical location and neurochemical phenotype of sensory neurons inside ICG. In the present study, rat ICG neurons were characterized neurochemically with immunohistochemistry using glutamatergic markers: vesicular glutamate transporters 1 and 2 (VGLUT1; VGLUT2), and glutaminase (GLS), the enzyme essential for glutamate production. Glutamatergic neurons (VGLUT1/VGLUT2/GLS) in the ICG that have axons to the ventricles were identified by retrograde tracing of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) injected in the ventricular wall. Co-labeling of VGLUT1, VGLUT2, and GLS with the vesicular acetylcholine transporter (VAChT) was used to evaluate the relationship between post-ganglionic autonomic neurons and glutamatergic neurons. Sequential labeling of VGLUT1 and VGLUT2 in adjacent tissue sections was used to evaluate the co-localization of VGLUT1 and VGLUT2 in ICG neurons. Our studies yielded the following results: (1) ICG contain glutamatergic neurons with GLS for glutamate production and VGLUT1 and 2 for transport of glutamate into synaptic vesicles; (2) atrial ICG contain neurons that project to ventricle walls and these neurons are glutamatergic; (3) many glutamatergic ICG neurons also were cholinergic, expressing VAChT; (4) VGLUT1 and VGLUT2 co-localization occurred in ICG neurons with variation of their protein expression level. Investigation of both glutamatergic and cholinergic ICG neurons could help in better understanding the function of the intrinsic cardiac nervous system.