Richard Kinkead

Role of endogenous opioid system in the regulation of the stress response

Numerous studies and reviews support an important contribution of endogenous opioid peptide systems in the mediation, modulation, and regulation of stress responses including endocrine (hypothalamopituitary-adrenal, HPA axis), autonomic nervous system (ANS axis), and behavioral responses. Although several discrepancies exist, the most consistent finding among such studies using different species and stressors is that opioids not only diminish stress-induced neuroendocrine and autonomic responses, but also stimulate these effector systems in the non-stressed state. A distinctive feature of the analgesic action of opioids is the blunting of the distressing, affective component of pain without dulling the sensation itself. Therefore, opioid peptides may diminish the impact of stress by attenuating an array of physiologic responses including emotional and affective states. The widespread distribution of enkephalin (ENK) throughout the limbic system (including the extended amygdala, cingulate cortex, entorhinal cortex, septum, hippocampus, and the hypothalamus) is consistent with a direct role in the modulation the stress responses. The predictability of stressful events reduces the impact of a wide range of stressors and ENK appears to play an important role in this process. Therefore, ENK and its receptors could represent a major modulatory system in the adaptation of an organism to stress, balancing the response that the stressor places on the central stress system with the potentially detrimental effects that a sustained stress may produce. Chronic neurogenic stressors will induce changes in specific components of the stress-induced ENKergic system, including ENK, delta- and mu-opioid receptors. This review presents evidences for adaptive cellular mechanisms underlying the response of the central stress system when assaulted by repeated psychogenic stress, and the involvement of ENK in these processes.

Neonatal maternal separation and sex-specific plasticity of the hypoxic ventilatory response in awake rat.

We tested the hypothesis that neonatal maternal separation (NMS), a form of stress that affects hypothalamo–pituitary–adrenal axis (HPA) function in adult rats, alters development of the respiratory control system. Pups subjected to NMS were placed in a temperature and humidity controlled incubator 3 h per day for 10 consecutive days (P3 to P12). Control pups were undisturbed. Once they reached adulthood (8–10 weeks old), rats were placed in a plethysmography chamber for measurement of ventilatory and cardiovascular parameters under normoxic and hypoxic conditions. Measurement of c‐fos mRNA expression in the paraventricular nucleus of the hypothalamus (PVH) combined with plasma ACTH and corticosterone levels confirmed that NMS effectively disrupted HPA axis function in males. In males, baseline minute ventilation was not affected by NMS. In contrast, NMS females show a greater resting minute ventilation due to a larger tidal volume. The hypoxic ventilatory response of male NMS rats was 25% greater than controls, owing mainly to an increase in tidal volume response. This augmentation of the hypoxic ventilatory response was sex‐specific also because NMS females show an attenuated minute ventilation increase. Baseline mean arterial blood pressure of male NMS rats was 20% higher than controls. NMS‐related hypertension was not significant in females. The mechanisms underlying sex‐specific disruption of cardio‐respiratory control in NMS rats are unknown but may be a consequence of the neuroendocrine disruption associated with NMS. These data indicate that exposure to a non‐respiratory stress during early life elicits significant plasticity of these homeostatic functions which persists until adulthood.

Human Metapneumovirus Infection Induces Long-Term Pulmonary Inflammation Associated with Airway Obstruction and Hyperresponsiveness in Mice

BACKGROUND Human metapneumovirus (hMPV) is a newly described paramyxovirus that is associated with bronchiolitis, pneumonia, and asthma exacerbation. The objective of the present work was to study the duration of pulmonary inflammation and the functional consequences of infection with hMPV by use of a BALB/c mouse model. METHODS BALB/c mice were inoculated with 1 x 10(8) TCID(50) of hMPV type A (C-85473), and viral persistence in lungs was assessed by reverse-transcription polymerase chain reaction for 154 days after infection. Pulmonary inflammation was characterized in histopathological experiments by use of a validated scoring system, and periodic acid-Schiff (PAS) staining of lung sections was used to document increased mucus production, also until day 154. Finally, respiratory functions were analyzed by taking plethysmographic measurements until day 70. RESULTS Persistence of viral RNA and significant pulmonary inflammation were noted until day 154, whereas the findings for PAS staining suggested that mucus production was increased only until day 12. Maximal breathing difficulties occurred on day 5, and airway obstruction and hyperresponsiveness were still significant until at least day 70. CONCLUSION Acute hMPV infection in BALB/c mice is associated with long-term pulmonary inflammation that leads to significant obstructive disease of the airways. This animal model will be of a great benefit in the evaluation of novel therapeutic and prophylactic modalities.

Effect of Chronic Psychogenic Stress Exposure on Enkephalin Neuronal Activity and Expression in the Rat Hypothalamic Paraventricular Nucleus

Abstract: This study tested the hypothesis that the activation pattern of enkephalinergic (ENKergic) neurons within the paraventricular nucleus of the hypothalamus (PVH) in response to psychogenic stress is identical whether in response to repeated exposure to the same stress (homotypic; immobilization) or to a novel stress (heterotypic; air jet puff). Rats were assigned to either acute or chronic immobilization stress paradigms (90 min/day for 1 or 10 days, respectively). The chronic group was then subjected to an additional 90‐min session of either heterotypic or homotypic stress. A single 90‐min stress session (immobilization or air jet) increased PVH‐ENK heteronuclear (hn) RNA expression. In chronically stressed rats, exposure to an additional stress session (whether homotypic or heterotypic) continued to stimulate ENK hnRNA expression. Acute immobilization caused a marked increase in the numbers of Fos‐immunoreactive and Fos‐ENK double‐labeled cells in the dorsal and ventral medial parvicellular, and lateral parvicellular subdivisions of the PVH. Chronic immobilization caused an attenuated Fos response (∼ 66%) to subsequent immobilization. In contrast, chronic immobilization did not impair ENKergic neuron activation within the PVH following homotypic or heterotypic stress. These results indicate that within the PVH, chronic psychogenic stress markedly attenuates the Fos response, whereas ENKergic neurons resist habituation, principally within the ventral neuroendocrine portion of the nucleus. This suggests an increase in ENK effect during chronic stress exposure. Homotypic (immobilization) and heterotypic (air jet) psychogenic stressors produce similar responses, including Fos, ENK‐Fos, and ENK hnRNA, within each subdivision of the PVH, suggesting similar processing for painless neurogenic stimuli.

Long-Term Consequences of Neonatal Caffeine on Ventilation, Occurrence of Apneas, and Hypercapnic Chemoreflex in Male and Female Rats

Caffeine is an adenosine receptor antagonist commonly used as a respiratory stimulant to treat neonatal apneas of premature newborn. Neonatal caffeine treatment (NCT) has long-term effects on adenosine receptor expression and distribution; however, the potential effects of NCT on respiratory control development are unknown. To address this issue, rat pups received orally each day from postnatal d 3–12, 15 mg/kg of caffeine (NCT), water (vehicle), or were undisturbed during early life (control). Measurements of resting ventilation, apnea index, and ventilatory response to moderate hypercapnia (Fico2 = 0.05) were made using whole-body plethysmography at postnatal d 20 (juvenile) and adulthood. At d 20, resting respiratory variables were not affected by the treatments. Juvenile NCT male rats showed a 22% higher minute ventilation response to hypercapnia than vehicle rats. However, oral gavage alone increased the frequency component of the response by 11%. In adult males, caffeine increased the resting respiratory frequency by 15%. In these animals, the tidal volume response to hypercapnia was increased by 15%, whereas the frequency response was decreased by 20%. In juvenile and adult females, no differences were observed between treatments. In juvenile rats of both sexes, gavage increased the apnea index by at least 200%. These results show that NCT and gavage influence respiratory control during early life and that these effects persist until adulthood. The underlying mechanisms are unclear, but may be related to persistent changes in adenosinergic neurotransmission because neonatal caffeine administration increases A1 adenosine receptor density in adult rats.

Neonatal maternal separation and enhancement of the hypoxic ventilatory response in rat: the role of GABAergic modulation within the paraventricular nucleus of the hypothalamus

Neonatal maternal separation (NMS) affects respiratory control development as adult male (but not female) rats previously subjected to NMS show a hypoxic ventilatory response 25% greater than controls. The paraventricular nucleus of the hypothalamus (PVN) is an important modulator of respiratory activity. In the present study, we hypothesized that in awake rats, altered GABAergic inhibition within the PVN contributes to the enhancement of hypoxic ventilatory response observed in rats previously subjected to NMS. During normoxia, the increase in minute ventilation following microinjection of bicuculline (1 mm) within the PVN is greater in NMS versus control rats. These data show that regulation of ventilatory activity related to tonic inhibition of the PVN is more important in NMS than control rats. Microinjection of GABA or muscimol (1 mm) attenuated the ventilatory response to hypoxia (12% O2) in NMS rats only. The higher efficiency of microinjections in NMS rats is supported by results from GABAA receptor autoradiography which revealed a 22% increase in GABAA receptor binding sites within the PVN of NMS rats versus controls. Despite this increase, however, NMS rats still show a larger hypoxic ventilatory response than controls, suggesting that within the PVN the larger number of GABAA receptors either compensate for (1) a deficient GABAergic modulation, (2) an increase in the efficacy of excitatory inputs converging onto this structure, or (3) both. Together, these results show that the life‐long consequences of NMS are far reaching as they can compromise the development of vital homeostatic function in a way that may predispose to respiratory disorders.

The Role of Circulating Catecholamines in the Ventilatory and Hypertensive Responses to Hypoxia in the Atlantic Cod (Gadus morhua)

This is the first description of the ventilatory response of the Atlantic cod (Gadus morhua) to hypoxia. Using a pharmacological approach, we tested the hypotheses that during hypoxia (1) elevation of circulating catecholamines, arising from chromaffin tissue or peripheral adrenergic neurones, contributes to hyperventilation and that (2) the hyperventilatory response can be modulated by the hypoxia-induced hypertension. The ventilatory response to hypoxia (final water PO₂ = 46 mmHg, reached in ≤25 min) was not significantly altered by pretreatment offish with either α-or β-adrenoceptor antagonists (phentolamine or sotalol, respectively) or with an inhibitor of catecholamine release from peripheral neurones (bretylium), despite a significant increase in plasma catecholamine levels in all treatment groups. Since the utilized regime did not induce metabolic acidosis (a potential ventilatory stimulant), we conclude that the hyperventilation was caused solely by a depression of the oxygen status. Although changes in the internal as well as the external oxygen status may have potentially contributed to the hyperventilation, we suggest, on the basis of the rapid ventilatory response after only small depressions of water PO₂, that the initial stimulus is external. During hypoxia, a doubling of mean ventral aortic blood pressure was observed. This elevation of blood pressure was due to an increased adrenergic nervous and humoral activity. The lack of modification in the ventilatory response to hypoxia after transient or persistent abolishment of the hypertension by pharmacological agents (bretylium or phentolamine, respectively) indicated that during hypoxia there was no relationship between blood pressure and ventilation in this species.

Neonatal maternal separation and early life programming of the hypoxic ventilatory response in rats.

The neonatal period is critical for central nervous system (CNS) development. Recent studies have shown that this basic neurobiological principle also applies to the neural circuits regulating respiratory activity as exposure to excessive or insufficient chemosensory stimuli during early life can have long-lasting consequences on the performance of this vital system. Although the tactile, olfactory, and auditory stimuli that the mother provides to her offspring during the neonatal period are not directly relevant to respiratory homeostasis, they likely contribute to respiratory control development. This review outlines the rationale for the link between maternal stimuli and programming of the hypoxic ventilatory response during early life, and presents recent results obtained in rats indicating that experimental disruption of mother-pup interaction during this critical period elicits significant phenotypic plasticity of the hypoxic ventilatory response.

Early-life risk factors for panic and separation anxiety disorder: Insights and outstanding questions arising from human and animal studies of CO2 sensitivity

Genetically informative studies showed that genetic and environmental risk factors act and interact to influence liability to (a) panic disorder, (b) its childhood precursor separation anxiety disorder, and (c) heightened sensitivity to CO2, an endophenotype common to both disorders. Childhood adversities including parental loss influence both panic disorder and CO2 hypersensitivity. However, childhood parental loss and separation anxiety disorder are weakly correlated in humans, suggesting the presence of alternative pathways of risk. The transferability of tests that assess CO2 sensitivity - an interspecific quantitative trait common to all mammals - to the animal laboratory setting allowed for environmentally controlled studies of early parental separation. Animal findings paralleled those of human studies, in that different forms of early maternal separation in mice and rats evoked heightened CO2 sensitivity; in mice, this could be explained by gene-by-environment interactional mechanisms. While several questions and issues (including obvious divergences between humans and rodents) remain open, parallel investigations by contemporary molecular genetic tools of (1) human longitudinal cohorts and (2) animals in controlled laboratory settings, can help elucidate the mechanisms beyond these phenomena.

Chronic corticosterone elevation and sex‐specific augmentation of the hypoxic ventilatory response in awake rats

Perinatal stress disrupts normal development of the hypothalamo‐pituitary‐adrenal (HPA) axis. Adult male (but not female) rats previously subjected to a stress such as neonatal maternal separation (NMS) are characterized by chronic elevation of plasma corticosterone (Cort) levels and an abnormally elevated hypoxic ventilatory response through mechanisms that remain unknown. The present study tested the hypothesis that a chronic increase of plasma Cort levels alone augments the ventilatory response to hypoxia in adult rats. Three groups of Sprague–Dawley male and female rats were used (control, placebo and Cort implants). Rats subjected to chronic Cort elevation received a subcutaneous Cort implant (300 mg) 14 days prior to ventilatory measurements, whereas sham‐operated rats received placebo implants. Controls received no treatment. Plasma Cort levels and body weight profiles were measured to assess protocol efficiency. Whole body plethysmography was used to measure ventilatory activity and metabolic indices during normoxia and following a 20 min period of moderate hypoxia (12% O2). Male rats implanted with Cort showed a ventilatory response to hypoxia higher than placebo‐treated rats; this effect was mainly due to a larger tidal volume response. In females, Cort treatment increased the breathing frequency response but the effect on minute ventilation was not significant. Taken together, these data show that chronic elevation of Cort alone increases the ventilatory response to hypoxia, but in a sex‐specific manner. These data raise important questions regarding the mechanisms underlying the sexual dimorphism of this effect and the potential link between HPA axis dysfunction and respiratory disorders related to abnormal ventilatory chemoreflex.