Sylvie Laforest

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.

Brief exposure to predator odor and resultant anxiety enhances mesocorticolimbic activity and enkephalin expression in CD-1 mice

The present study assessed alterations in mesolimbic enkephalin (ENK) mRNA levels after predator [2,5‐dihydro‐2,4,5‐trimethylethiazoline (TMT)] and non‐predator (butyric acid) odor encounter and/or light–dark (LD) testing in CD‐1 mice immediately, 24, 48 and 168 h after the initial odor encounter and/or LD testing. The nucleus accumbens, ventral tegmental area, basolateral (BLA), central (CEA) and medial amygdaloid nuclei, prelimbic and infralimbic cortex were assessed for fos‐related antigen (FRA) and/or ENK mRNA as well as neuronal activation of ENK neurons (FRA/ENK). Mice exposed to TMT displayed enhanced freezing and spent less time in the light of the immediate LD test relative to saline‐ or butyric acid‐treated mice. Among mice exposed to TMT, LD anxiety‐like behavior was associated with increased FRA in the prelimbic cortex and accumbal shell and decreased ENK‐positive neurons in the accumbal core. Mice displaying high TMT‐induced LD anxiety exhibited increased ENK‐positive neurons in the BLA, CEA and medial amygdaloid nuclei relative to mice that displayed low anxiety‐like behavior in the LD test after TMT exposure. In the BLA and CEA, ‘high‐anxiety’ mice also displayed increased FRA/ENK after TMT exposure and LD testing. In contrast to neural cell counts, the level of ENK transcript was decreased in the BLA and CEA of ‘high‐anxiety’ mice after TMT exposure and LD testing. These data suggest that increased FRA may regulate stressor‐responsive genes and mediate long‐term behavioral changes. Indeed, increased ENK availability in mesolimbic sites may promote behavioral responses that detract from the aversiveness of the stressor experience.

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.

Enkephalinergic Afferents of the Centromedial Amygdala in the Rat

The connectivity of the amygdaloid complex has been extensively explored with both anterograde and retrograde tracers. Even though the afferents of the centromedial amygdala [comprising the central (CEA) and medial (MEA) amygdaloid nuclei] are well established, relatively little is known about the neuropeptide phenotype of these connections. In this study, we first examined the distribution of μ‐opioid receptor (MOR) and δ‐opioid receptor (DOR) in the amygdala via in situ hybridization and immunohistochemistry. We then investigated the distribution of Met‐enkephalin (ENK) and Leu‐ENK fibers with immunohistochemistry and examined the distribution of preproenkephalin mRNA in the amygdala by using in situ hybridization. Finally, we examined the ENK projections to the CEA and MEA by using stereotaxic injections of the retrograde tracer cholera toxin subunit B or fluorogold revealed by immunohistochemistry combined with in situ hybridization to identify ENKergic neurons. Our results indicate that the centromedial amygdala receives ENK afferents, as indicated by the presence of MOR, DOR, and ENK fibers in the CEA and MEA, originating primarily from the bed nucleus of the stria terminalis (BST) and from other amygdaloid nuclei. The posterior BST, the basomedial nucleus (BMA), and the cortical nucleus of the amygdala (COA) were found to be the major ENK afferents of the MEA, whereas the anterolateral BST, the COA, the MEA, and the BMA provided the main ENKergic innervation of the CEA. In addition, we found that the ventromedial nucleus of the hypothalamus and the pontine parabrachial nucleus provide a moderate ENK input to the CEA and MEA. The functional implications of these connections in stress, anxiety, and nociception are discussed. J. Comp. Neurol. 496:859–876, 2006.

Changes in brain cholecystokinin and anxiety-like behavior following exposure of mice to predator odor

Exposure of CD-1 mice to a familiar environment lined with clean shavings (control odor) as well as a familiar environment lined with soiled rat shavings (predator odor) induced anxiety in the light/dark box. Mice exposed to the familiar environment or predator odor displayed decreased latency to enter the dark chamber of the light/dark box and spent less time in the light portion of the apparatus relative to home-caged mice. Mice exposed to the familiar environment lined with clean shavings or predator odor displayed elevated cholecystokinin mRNA levels from the ventral tegmental area, medial and basolateral nuclei of the amygdala relative to home-caged mice. Exposure of CD-1 mice to 2, 5 or 10 min of predator odor increased acoustic startle relative to mice merely exposed to the familiar environment lined with clean shavings at protracted intervals. Mice exposed to the familiar environment lined with clean shavings did not exhibit enhanced startle relative to home-caged mice. Exaggerated startle reactivity was in evidence immediately, 24, and 48 h following a 5-min exposure of mice to predator odor. In contrast, a 10-min exposure of mice to predator odor produced an oscillating pattern of enhanced startle evident during the immediate and 48-h post-stressor intervals only. However, when the startle stimulus was withheld 1 h following odor presentation, mice exhibited enhanced startle patterns reminiscent of the 5-min exposure. The 2-min exposure of mice to predator odor produced a delayed onset of enhanced startle observed at the 168-h test interval only. Potential anxiogenic influences of mesocorticolimbic cholecystokinin availability as well as the time course and underlying neuronal substrates of long-term behavioral disturbances as a result of psychogenic stressor manipulations are discussed.

Involvement of central angiotensin receptors in stress adaptation

The present study examined the effects of acute and chronic neurogenic stressors on the expression of two distinct angiotensin receptors in two stress-related brain nuclei: angiotensin type 1A receptor in the paraventricular nucleus of the hypothalamus and angiotensin type 2 receptor in the nucleus locus coeruleus. Male Wistar rats were divided into four experimental groups. The first two groups were subjected once to an acute 90-min immobilization or air-jet stress session, respectively. The other two groups were subjected to 10 days of daily 90-min immobilization sessions and, on the 11th day, one group was exposed to an additional 90-min immobilization and the other to a single air-jet stress (heterotypic but still neurogenic) session. In each group, rats were perfused before stress (0 min), immediately following stress (90 min) or 150, 180, 270 or 360 min (and 24 h in chronic immobilization) after the beginning of the last stress session. Basal expression of both angiotensin receptor subtype 1A and angiotensin receptor subtype 2 messenger RNA was minimal in non-stressed animals. Acute immobilization as well as air-jet stress induced similar patterns (time-course and maximal values) of angiotensin receptor subtype 1A messenger RNA expression in the paraventricular nucleus. Angiotensin receptor subtype 1A messenger RNA expression increased 90-150 min after the beginning of the stress and returned to basal levels by 360 min. Chronic stress immobilization slightly modified the pattern, but not maximal values of angiotensin receptor subtype 1A messenger RNA expression to further immobilization (homotypic) or air-jet stress (heterotypic). Acute immobilization and air-jet stress sessions induced similar locus coeruleus-specific angiotensin receptor subtype 2 messenger RNA expression. This expression increased 90 min following the onset of the stress session and remained elevated for at least 360 min. Chronic immobilization stress increased angiotensin receptor subtype 2 messenger RNA expression to levels comparable to those observed in acute stress conditions. Novel acute exposure to neurogenic stressors did not further increase these levels in either homotypic (immobilization) or in heterotypic (air-jet stress) conditions. These results suggest that central angiotensin receptors are targets of regulation in stress; therefore, stress may modulate angiotensin function in the paraventricular nucleus and locus coeruleus during chronic exposure to neurogenic stressors.

Odor-Induced Variation in Anxiety-Like Behavior in Mice is Associated with Discrete and Differential Effects on Mesocorticolimbic Cholecystokinin mRNA Expression

The present investigation assessed alterations in mesocorticolimbic cholecystokinin (CCK) mRNA following novel predator and non-predator odor exposure and light-dark testing in CD-1 mice. In brief, acute exposure of CD-1 mice to the predator odor, 2,5-dihydro-2, 4,5-trimethylthiazoline (TMT; the major component of the anal gland secretions of the red fox), or the control odor, butyric acid (BA), suppressed rearing behavior during odor presentation, subsequently induced anxiety in the light dark test, and was associated with increased mesocorticolimbic CCK mRNA relative to saline treated mice. Only mice exposed to TMT displayed elevated freezing behaviors during odor treatment. In the light-dark test, mice exposed to either BA or TMT took longer to reenter the light section of the apparatus and spent less cumulative time in the light relative to mice exposed to saline. The decreased time spent in the light as well as light dark transitions were exaggerated among mice exposed to fox odor. Odor presentation was associated with increased CCK mRNA in mesocorticolimbic sites. Butyric acid was associated with enhanced CCK gene expression in the VTA, while both BA and TMT were associated with increased medial prefrontal cortex (mPFC) CCK mRNA levels. Increased CCK mRNA within the VTA and mPFC was evident among mice despite testing in the light-dark box. In contrast, basolateral nucleus of the amygdala (BLA) CCK mRNA was enhanced following odor exposure among mice in the light dark test relative only to saline treated mice which demonstrated a natural decrease in BLA CCK mRNA following the light dark test. The differential pattern of CCK mRNA associated with discrete psychogenic stressor manipulations and the provocation of anxiety-like behavior associated with such experiences is discussed.

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.

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.

Acute sodium deficit triggers plasticity of the brain angiotensin type 1 receptors

The brain renin‐angiotensin system (bRAS) is involved in the control of hydromineral balance. However, little information is available on the functional regulation of the bRAS as a consequence of sodium deficit in the extracellular fluid compartments. We used a pharmacological model of acute Na+ depletion (furosemide injections) to investigate changes of a major component of the bRAS, the hypothalamic angiotensin type 1A (AT1A) receptors. Furosemide induced a rapid and long‐lasting expression of the AT1A mRNA in the subfornical organ, the median preoptic nucleus (MnPO), and the parvocellular division of the paraventricular nucleus (pPVN). Na+ depletion increased the number of cells expressing AT1A mRNA in the pPVN, but not in the MnPO. The enhancement of AT1A mRNA expression was associated with an increase in AT1 binding sites in all the regions studied. It is of interest that in the paraventricular nucleus, the majority of the neurons expressing AT1A mRNA also showed an increase in metabolic activity (Fos‐related antigen immunoreactivity [FRA‐ir]). By contrast, in the MnPO, we observe two distinct cell populations. Our data demonstrated that an acute Na+ deficit induced a functional regulation of the hypothalamic AT1A receptors, indicating that these receptors are subject to plasticity in response to hydromineral perturbations.