Dissecting adrenal and behavioral responses to stress by targeted gene inactivation in mice

To define the molecular pathways modulating adrenal and behavioral responses to stress, we have generated mice with inactivation of hypothalamic neuropeptides and signaling pathways. Studies in mice deficient in corticotropin-releasing hormone (CRH) have revealed the essential role for CRH in adrenal glucocorticoid production in response to many physiological and psychological stressors. Immune system activation in CRH-deficient mice provides a unique exception to the necessity for CRH in stimulating adrenal glucocorticoid production. By analyzing mice deficient in interleukin-6 (IL-6) and CRH, we find that restoration of glucocorticoid output with inflammation is largely mediated by dysregulated IL-6 production. Current studies focus on identifying cellular and gene targets by which glucocorticoids regulate immune system function. In contrast to impaired adrenocortical responses to stress, CRH-deficient mice exhibit normal behavioral responses to stress. To determine signaling pathways that may contribute to the behavioral responses to stress, we have generated and analyzed mice deficient in adenylyl cyclase type 8 (AC8). AC8 deficient mice have intact adrenocortical responses to stress, but an inability to undergo stress-induced alterations in behavior.     

背景应激对静息态下大脑自发神经活动的影响

本研究试图探究背景应激对应激相关大脑自发神经活动的影响。使用日常应激水平作为背景应激的量化指标。同时，使用静息态fMRI技术，采集了个体在静息态下的大脑自发神经活动，并使用局部一致性（ReHo）作为指标。结果发现，背景应激越高的个体，右侧海马（扩展到丘脑和脑干）静息态下局部一致性水平越高。结果提示，经历了高背景应激的个体会表现出边缘系统脑区的自发神经活动的持续活跃状态。     

Cytokine production by spleen cells after social defeat in mice: activation of T cells and reduced inhibition by glucocorticoids

Social disruption (SDR) is an effective model of social stress associated with an enhanced inflammatory reactivity of the immune system. The aim of the present study was to further describe SDR effects on cytokine production by spleen cells, testing selectively monocyte and T cell functions as a result of this stressor. For this purpose, splenocytes from control mice (C) and mice socially stressed for 7 days (SDR) were cultured in the presence of lipopolysaccharide (LPS) or concanavalin A (Con A). Splenocyte proliferation, cytokine production and sensitivity of spleen cells to corticosterone were assessed in vitro. The humoral response to keyhole limpet hemocyanin (KLH) immunization was assessed. SDR induced splenomegaly and enhanced splenocyte basal proliferation. The pro-inflammatory influence of SDR was confirmed by an increased release of interleukin-6 (IL-6) by LPS-stimulated cultures and by a reduced sensitivity of spleen cells to the anti-inflammatory effect of corticosterone. The mechanism increasing cytokine production in response to LPS was cytokine specific, since among inflammatory cytokines, IL-6 but not interferon-gamma (IFN-gamma) was enhanced by stress. In stressed mice, the increase in IL-6 and IFN-gamma and the decrease in IL-10 release in Con A-stimulated cultures indicate that SDR did not modify the Th1/Th2 cytokine balance but globally activated T cells. Plasma anti-KLH antibody levels were similar in both groups. Wounded and non-wounded mice presented similar responses to stress. This study shows that social disruption stress enhances the reactivity of cells from both the acquired and innate immune systems.     

Epigenetic Alterations Associated with War Trauma and Childhood Maltreatment

Survivors of war trauma or childhood maltreatment are at increased risk for trauma-spectrum disorders such as post-traumatic stress disorder (PTSD). In addition, traumatic stress has been associated with alterations in the neuroendocrine and the immune system, enhancing the risk for physical diseases. Traumatic experiences might even affect psychological as well as biological parameters in the next generation, i.e. traumatic stress might have transgenerational effects. This article outlines how epigenetic processes, which represent a pivotal biological mechanism for dynamic adaptation to environmental challenges, might contribute to the explanation of the long-lasting and transgenerational effects of trauma. In particular, epigenetic alterations in genes regulating the hypothalamus–pituitary–adrenal axis as well as the immune system have been observed in survivors of childhood and adult trauma. These changes could result in enduring alterations of the stress response as well as the physical health risk. Furthermore, the effects of parental trauma could be transmitted to the next generation by parental distress and the pre- and postnatal environment, as well as by epigenetic marks transmitted via the germline. While epigenetic research has a high potential of advancing our understanding of the consequences of trauma, the findings have to be interpreted with caution, as epigenetics only represent one piece of a complex puzzle of interacting biological and environmental factors. Copyright © 2015 John Wiley & Sons, Ltd.     

Stress, emotion, and human immune function

This article provides a review of empirical evidence linking emotional processes to immune function in humans. Acute stressors have produced mixed effects on immunity, presumably through differential activation of physiological stress systems. Chronic stress has been associated with suppression of immune function, and there is evidence that the immune system may not adapt over time. Effects of stress accompanying social disruption and psychological depression, when demonstrated, have been consistently adverse. Certain personality styles may enhance or degrade immune response. Relationships between psychosocial factors and immunity have been identified for several diseases, including cancer, acquired immune deficiency syndrome, and autoimmune diseases; psychosocial interventions have been tested with variable results. Theoretical and methodological considerations are summarized and directions for future research suggested.     

不同时程应激对大鼠行为、免疫和交感神经系统反应的影响

研究旨在研究不同时程情绪应激诱导的大鼠行为、免疫和神经内分泌反应的动态变化及其相互关系。实验采用一种在程序性饮水的固定时间点不确定性给予大鼠空瓶刺激诱发其情绪反应的应激模式,测定不同时程（14,21和28天）应激对大鼠行为、交感神经系统反应、体液免疫功能及体重增长的影响。结果表明,情绪应激导致大鼠攻击行为显著增加,且在整个应激过程中情绪应激组大鼠的攻击行为都被稳定地诱导;不同时程的情绪应激均导致大鼠特异性抗OVA抗体水平明显降低,且降低的程度类似;在情绪应激的第14天和28天应激组大鼠血中去甲肾上腺素水平明显高于对照组,但后者升高的水平明显低于前者。此外,14天应激明显抑制大鼠的体重增长,但随着应激时程的延长,体重增长逐渐恢复。这些结果表明,随着应激时程的延长,大鼠行为、免疫和神经内分泌反应的适应性改变存在时程差异,有助于进一步了解应激诱发的各种反应间的复杂关系     

Influence of the specific immune response on some consistent murine behaviors.

The author's goal was to discover if the generation and maintenance of the specific immune response resulted in alterations of reliable behaviors (i.e., behaviors correlated over time). The behaviors (ambulation, rearing, and interaction with a conspecific) of CD1 male mice were measured in a small open field, and several days later, the mice were immunized with antigens (either splenocytes from C57BL/6 mice or a mixture of sheep erythrocytes and goat serum). The same behaviors were recorded again some hours, or some days, after immunization. Immunizations and behavioral measurements were repeated at various intervals. Blood levels of antibodies to the antigens were measured 6 days after immunization. The recorded behaviors were consistent (according to Kendall coefficient of concordance). The mice mounted antibody responses to the antigens, yet no behavioral changes were apparent during the response. On the contrary, a single injection of E. coli lipopolysaccharide decreased ambulation and rearing. It is proposed that in healthy mice kept in normal conditions, the specific immune response may be unrelated to reliable behavioral changes.     

Bridging psychology and biology. The analysis of individuals in groups

Biological systems are particularly prone to variation, and the authors argue that such variation must be regarded as important data in its own right. The authors describe a method in which individual differences are studied within the framework of a general theory of the population as a whole and illustrate how this method can be used to address three types of issues: the nature of the mechanisms that give rise to a specific ability, such as mental imagery; the role of psychological or biological mediators of environmental challenges, such as the biological bases for differences in dispositional mood; and the existence of processes that have nonadditive effects with behavioral and physiological variables, such as factors that modulate the response to stress and its effects on the immune response.     

The effects of instructional set on physiological response to stressful imagery

Centrally mediated or cognitive variables have received considerable attention in clinical research. With the establishment of the effectiveness of such specific treatment techniques as systematic desensitization (Paul. 1969a. 1969b). a question arises as to the influence of cognitions on process and outcome variables in such learning based treatments (Lang. 1971). Research investigating the influence of demand characteristics and subject expectancies has demonstrated such centrally mediated variables can significantly influence overt, behavioral and self-report measures of fear, stress, or anxiety (cf. Borkovec. 1972: Marcia. Rubin and Efran. 1969; McGlynn. Maelia and Nawas. 1969: McGlynn. Reynolds and Linder. 1971: Oliveau. Agras. Leitenberg and Wright. 1969; Rappaport. 1972: Rosen. 1974).However. Borkovec (1972) and Rappaport (1972) failed to obtain expectancy effects on physiological measures of anxiety. Neither investigation provided for an independent assessment of subjects' actual expectancies, or belief in the instructions, as recommended by Davison and Wilson (1973). Borkovec administered clinical procedures over four sessions and varied the information communicated about the ‘purpose’ of the treatments (physiological versus therapeutic instructions). Differential rationales for differential expectancies of physiological reactivity were not provided. After each session, subjects reviewed the same false physiological records depicting reductions in fear responses. Although these changes were explained either physiologically or therapeutically. the fact that such reductions were emphasized should have attenuated differences between groups. Rappaport (1972) exposed subjects to a fearful stimulus and varied the suggestion and rationale for the pseudotreatment: no expectancy (stress research): therapeutic expectancy (fear reduction); and negative expectancy (fear increase). The lack of an independent assessment of the expectancy manipulation, as well as the exclusion of a specific therapeutic procedure, make it impossible to interpret the results in relation to expectancy effects on physiological fear responses during the administration of therapeutic procedures. There is other evidence, however, that suggestion and instructional set can significantly influence somatic and autonomic response systems (Barber. 1961. 1965; Sternbach. 1964. 1965).The primary purpose of this study was to evaluate the effects of instructional set on physiological responses to stressful imagery. This was an early step in a research program investigating the effects of instructional and informational variables on physiological responses of clinical interest. One half of the subjects received abbreviated live relaxation training (R) as described m a manual by Bernstein and Borkovec (1972) and based upon the original work of Jacobson (1938). The other half received an inert placebo pill and undertook a target detection task (P). The latter procedure was similar to that used by Paul (1966). Although both procedures had previously been shown to have relaxing effects, the P task was included as an additional control for any potential effects specific to the tension-release procedure of R. In order to evaluate the effects of instructional set, one half of each of the above two groups received instructions designed to lead to an expectancy of response inhibition to stressful imagery by the respective procedure (Decrease Stress Response. DSR). while the other half received instructions designed to lead to an expectancy of an increased response to stressful imagery by the respective procedure (Increase Stress Response. ISR). Each subject visualized three individually specified items that were the most frightening scenes imaginable prior to training and four times after training visualized the scene producing the largest response. Considerable effort was devoted to eliciting from each subject the most frightening items possible for her. and it was clear from subject comments that the items did elicit negative emotional responses.Since previous research (Barber. 1961. 1965; Sternbach. 1964. 1965) indicated that instructional set can influence physiological reactivity, it was hypothesized the DSR) subjects should exhibit a greater reduction in emotional response from pre- to posttraining than ISR subjects.     

Effects of maternal stress on development and behaviour in rat offspring

Retrospective studies suggest that gestational stress in humans can delay the attainment of developmental milestones, increase the incidence of allergic reactions and respiratory infections and cause behavioural abnormalities in the children. Our studies and others have shown that prenatal stress in rats can mimic several of these developmental and behavioural alterations. These include a suppression of immune function, but also enhanced sensitivity to allergens. Prenatally-stressed rats, like children, show a reduced propensity for social interaction and increased anxiety in intimidating or novel situations. They have physiological and behavioural alterations consistent with depressive symptoms, including a phase-shift in their circadian rhythm for corticosterone, sleep abnormalities, and greater acquisition of learned helplessness under appropriate conditions. Prenatally-stressed male rats also show demasculinisation and feminisation of their sexual behaviour. The developmental and behavioural abnormalities in prenatally-stressed offspring may be mediated by alterations in the activity of endogenous opioids or neurosteroids, since several of them can be corrected by maternal administration of an opioid antagonist or by drugs like diazepam and allopregnanolone that modulate GABA transmission.     

Motivational factors in health and disease

Evidence is reviewed that measures of motive strength, as measured through content analysis of associative thought, are related to physiological systems, the functioning of which affect health outcomes. Studies show that affiliative and power motive syndromes assessed in associative thought are associated with health and illness. A relaxed or easygoing affiliative motive syndrome characterizes insulin dependent Type I diabetics and can, if aroused, lead to poorer blood sugar control in such diabetics. A stressed power motive syndrome is associated with sympathetic activation, release of stress hormones, depressed immune functions, and greater susceptibility to infectious diseases. Affiliative trust and a greater sense of agency as measured in associative thought content are associated with better health. An intervention study and a longitudinal study have demonstrated that differences in the levels of these motivational variables are not simply the result of illness but lead to alterations in subsequent health status.     

Heart rate responses to induced challenge situations in greylag geese (Anser anser)

Adequate short-term responses to stressors are of great importance for the health and well-being of individuals and factors modulating the physiological stress response (e.g., controllability, suddenness, familiarity) of a stimulus are well described under laboratory conditions. In the present study we aimed at investigating the stress response in greylag geese (Anser anser) in the field, confronting individuals with naturally occurring stressors. We measured beat-to-beat heart rate (HR) via fully implanted transmitters during three different experimental challenges: (1) catching and holding, (2) confrontation with a model predator, and (3) approach by different humans. We compared this to a control period and HR during agonistic encounters, a naturally occurring stressor. All three experimental situations evoked a HR increase. Highest HR responses were elicited by catching and holding the animals. In the third experiment, HR responses were greatest when the geese were approached by a human stranger (i.e., somebody the geese have never seen before). Hence, geese discriminated between different kinds of stressors and adjusted their physiological response depending on the type of stressor. Our results show that geese were able to discriminate between individual humans. In line with a number of lab studies, we suggest that particularly the controllability of certain situation determines the intensity of the HR response, also in a natural setting in the field.     

Hemispheric asymmetry in stress processing in rat prefrontal cortex and the role of mesocortical dopamine

The prefrontal cortex (PFC) is known to play an important role not only in the regulation of emotion, but in the integration of affective states with appropriate modulation of autonomic and neuroendocrine stress regulatory systems. The present review highlights findings in the rat which helps to elucidate the complex nature of prefrontal involvement in emotion and stress regulation. The medial PFC is particularly important in this regard and while dorsomedial regions appear to play a suppressive role in such regulation, the ventromedial (particularly infralimbic) region appears to activate behavioral, neuroendocrine and sympathetic autonomic systems in response to stressful situations. This may be especially true of spontaneous stress-related behavior or physiological responses to relatively acute stressors. The role of the medial PFC is somewhat more complex in conditions involving learned adjustments to stressful situations, such as the extinction of conditioned fear responses, but it is clear that the medial PFC is important in incorporating stressful experience for future adaptive behavior. It is also suggested that mesocortical dopamine plays an important adaptive role in this region by preventing excessive behavioral and physiological stress reactivity. The rat brain shows substantial hemispheric specialization in many respects, and while the right PFC is normally dominant in the activation of stress-related systems, the left may play a role in countering this activation through processes of interhemispheric inhibition. This proposed basic template for the lateralization of stress regulatory systems is suggested to be associated with efficient stress and emotional self-regulation, and also to be shaped by both early postnatal experience and gender differences.     

The transactional psychobiological nature of cognitive appraisal during exercise in environmentally stressful conditions

Background and Purpose: Successful adaptation to the stress of physical exertion in adverse environmental conditions (heat, cold, high altitude) is of great concern when optimal performance within safe parameters is the goal. The perception of the psychophysical demands imposed by the stressful situation and the perceived capability to cope with these demands is a process that can dramatically alter the intensity of the ensuing physiological activation. Thus, exercise in environmentally stressful conditions provides an excellent model for examining the relationship between the cognitive appraisal of the physical stress and the ensuing stress response. A brief review of the research on cognitive appraisals during exercise in stressful environments provides evidence of the connection between cognitive appraisals and the stress response during exercise under environmental stress and demonstrates a need for a transactional psychobiological model proposed in this paper. This model attributes a central role to the continuous cognitive appraisal of the situation by the individual.Methods: Computer searches of psychological, sport science, and medical databases using the terms exercise, heat, cold, high altitude, environmental stress, and ratings of perceived exertion were conducted. Additionally, the reference citations in the obtained articles were searched for relevant studies. An abridged integrated review summarizes the critical findings and limitations. Furthermore, literature supporting the proposed transactional psychobiological model is presented.Results and Conclusions: Further investigation into the psychophysical and the affective responses to exercise in adverse environmental conditions can be facilitated through the utilization of the proposed transactional psychobiological model.     

Prenatal stress and risk for psychopathology: specific effects or induction of general susceptibility?

This review focuses on prenatal stress as a risk factor for psychopathology. Evidence from animal studies is summarized, and the relevance of prenatal stress models in animals for human studies is discussed. In the offspring of prenatally stressed animals, overactivity and impaired negative feedback regulation of the hypothalamic-pituitary-adrenal axis are consistent findings and may reflect a pathophysiological mechanism involved in the development of psychopathology. Reduced activity of the opioid GABA/benzodiazepine, serotonin, and dopamine systems and increased activity of the sympathico-adrenal system have been found as well. These alterations have been linked to a diverse spectrum of psychopathology. Therefore, the evidence supports the view that exposure to prenatal stress may result in a general susceptibility to psychopathology, rather than exerting a direct effect on a specific form of psychopathology.     

The impact of an emotional self-management skills course on psychosocial functioning and autonomic recovery to stress in middle school children

Unmanaged emotional reactions to stress not only lead to behavior problems in young people but also create physiological conditions that inhibit learning and potentially increase the risk of disease later in life. For these reasons, the integration of emotional self-management skills training programs has become an increased priority in some schools. In this study, middle school students enrolled in a course in emotional competence skills learned techniques designed to intercept stressful responses during emotionally challenging situations. Behavioral outcomes were assessed using the Achievement Inventory Measure and autonomic function was measured by heart rate variability (HRV) analysis during and after a stressful interview. Following the program, students exhibited significant improvements in areas including stress and anger management, risky behavior, work management and focus, and relationships with family, peers and teachers. These improvements were sustained over the following six months. Students using the skills taught in the course to recover from acute emotional stress were also able to positively modulate their physiological stress responses. As compared to a control group, trained students demonstrated significantly increased HRV and more rhythmic, sine wave-like heart rhythm patterns during recovery. This response pattern reflects increased parasympathetic activity, heart rhythm coherence, and entrainment of other biological oscillatory systems to the primary heart rhythm frequency. Increased physiological coherence is associated with improved cognitive performance, emotional balance, mental clarity and health outcomes. These physiological shifts could promote the sustained psychological and behavioral improvements associated with the use of emotional management skills. It is suggested that learning emotional competence skills in childhood establishes healthier physiological response patterns which can benefit learning and long-term health. Results provide support for the integration in school curricula of courses designed to teach effective self-management skills to children.     

Endocannabinoids and stress

Endogenous cannabinoids play an important role in the physiology and behavioral expression of stress responses. Activation of the hypothalamic-pituitary-adrenal (HPA) axis, including the release of glucocorticoids, is the fundamental hormonal response to stress. Endocannabinoid (eCB) signaling serves to maintain HPA-axis homeostasis, by buffering basal activity as well as by mediating glucocorticoid fast feedback mechanisms. Following chronic stressor exposure, eCBs are also involved in physiological and behavioral habituation processes. Behavioral consequences of stress include fear and stress-induced anxiety as well as memory formation in the context of stress, involving contextual fear conditioning and inhibitory avoidance learning. Chronic stress can also lead to depression-like symptoms. Prominent in these behavioral stress responses is the interaction between eCBs and the HPA-axis. Future directions may differentiate among eCB signaling within various brain structures/neuronal subpopulations as well as between the distinct roles of the endogenous cannabinoid ligands. Investigation into the role of the eCB system in allostatic states and recovery processes may give insight into possible therapeutic manipulations of the system in treating chronic stress-related conditions in humans.     

Connection between the evaluation of positive or negative valence and verbal responses to a lexical decision making task]

Evaluation of the positive or negative valence of a stimulus is an activity that is part of any emotional experience that has been mostly studied using the affective priming paradigm. When the prime and the target have the same valence (e.g. positive prime and positive target), the target response is facilitated as a function of opposing valence conditions (e.g. negative prime and positive target). These studies show that this evaluation is automatic but depends on the nature of the task's implied response because the priming effects are only observed for positive responses, not for negative responses. This result was explained in automatic judgmental tendency model put forth by Abelson and Rosenberg (1958) and Klauer and Stern (1992). In this model, affective priming assumes there is an overlap between both responses, the first response taking precedence as a function of the prime-target valence, and the second response one that is required by the task. We are assuming that another type of response was not foreseen under this model. In fact, upon activating the valence for each of the prime-target elements, two preliminary responses would be activated before the response on the prime-target valence relationship. These responses are directly linked to the prime and target evaluation independently of the prime-target relationship. This hypothesis can be linked to the larger hypothesis whereby the evaluative process is related to two distinct motivational systems corresponding to approach and avoidance behaviour responses (Lang, Bradley, & Cuthbert, 1990; Neuman & Strack, 2000; Cacciopo, Piester & Bernston, 1993). In this study, we use the hypothesis that when a word leads to a positive valence evaluation, this favours a positive verbal response and inversely, a negative valence word favours a negative response. We are testing this hypothesis outside the affective priming paradigm to study to what extent evaluating a word, even when it is not primed, activates both motivational systems and consequently, positive verbal responses for approach and negative responses for avoidance. To validate this hypothesis, we are re-using both versions of the lexical decision task proposed by Wentura (2000). The classic version leads participants to a positive response for words, and the modified version leads to a no response. This experiment, carried out with thirty-two participants, measures the influence on response time of two experimental factors, the intrasubject valence of words (positive and negative) and the inter-subject factor (yes and no responses to words). Results show an interaction between the type of response and word valence. It is temporally more onerous to give a no response to positive words than to negative words. This result confirms that there is a direct relation between the evaluation of a valence stimulus and the response to this stimulus, a relation that had up to now been essentially observed with motor behaviours, and more rarely with verbal responses. We propose integrating the existence of this link between evaluation and verbal response (yes and no) in interpreting the effects of affective priming.     

ROMANTIC PARTNERS' COPING STRATEGIES AND PATTERNS OF CORTISOL REACTIVITY AND RECOVERY IN RESPONSE TO RELATIONSHIP CONFLICT

Stress in close relationships can have significant negative consequences for mental health, physical health, and long-term relationship functioning. Dysregulated physiological responses to stress are potential pathways through which relationship stress may lead to these kinds of outcomes, and the ways in which individuals attempt to cope with relationship stress are likely to impact their physiological responses. However, our understanding of the specific coping strategies that predict physiological reactivity and recovery in these contexts is rather limited. This study explored relations between young adult college students' self-reported methods of coping with stress in their romantic relationships and their physiological reactivity to and recovery from negotiating conflict with their romantic partners. Partners' coping styles were also examined as predictors of physiological stress responses. One hundred and ninety opposite-sex couples (N = 380; modal length of relationship = 1-2 years) participated in an experimental conflict discussion task. Physiological stress reactivity to the task was assessed using salivary cortisol, a primary hormonal product of the hypothalamic-pituitary-adrenocortical (HPA) axis. Growth modeling of the cortisol levels before, during, and after the conflict task indicated that men who typically coped with relationship stress by seeking social support showed greater physiological reactivity to the conflict task. Partners' need for social support predicted stronger stress responses for both men and women, as well. While seeking social support is generally thought to be an adaptive coping strategy for couples, the results suggest that within the context of conflict negotiation in which receiving and providing support may be more difficult, seeking support from a partner is associated with greater phyisological stress.     

Physiological markers of traffic-related stress during active travel

Understanding perceptions of safety and comfort (PSC) while walking or cycling is essential to accommodating and encouraging active travel, but current measures of PSC, primarily surveys, suffer from validity and reliability issues. Physiological markers of stress like electrodermal activity and heart rate variability have been proposed as alternative, objective measures of PSC. This paper presents a literature summary and conceptual framework examining the use of physiological stress markers during walking and cycling. The existing studies of active traveller stress markers report inconsistent findings and account for limited controls. We propose a comprehensive conceptual framework to describe the array of dynamic stimuli experienced during active travel, with complex appraisals and multidimensional stress responses that feedback to travel behaviour and stimuli exposure, and culminate in a set of physiological outcomes triggered by activation of the autonomic nervous system – all moderated by numerous personal and trip-related factors. The key challenge of inferring traffic-related fear or discomfort from physiological markers measured on-road is potential confounding effects of: (1) non-traffic factors that induce or modify stress responses, (2) traffic factors that induce stress responses not associated with safety or comfort, and (3) personal and environmental factors that directly influence physiological measurements outside of a stress response. No physiological stress marker has yet been shown to be reliable for on-road active travellers, particularly not for inter-subject comparisons. Physiological markers have the potential to provide high-resolution, objective information about pedestrian and cyclist PSC, but further research, particularly controlled experiments, and more precise study framing are needed to ensure validity and address moderating and confounding factors.