Shimon Amir

The role of endorphins in stress: Evidence and speculations ☆

Several lines of evidence suggest that the endogenous opioid peptides endorphins may play a role in the defensive response of the organism to stress. The present paper summarizes these findings as well as evidence linking endorphins to the anterior pituitary polypeptide hormone adrenocorticotropin (ACTH). Evidence is presented that endorphins may function as trophic hormones in peripheral target organs such as the adrenal medulla and the pancreas. As such they may be part of the physiological mechanisms that mediate adrenaline and glucagon release in response to stress. Endorphins (enkephalins) are also suggested to play a role in the control of the pituitary gland during stress. In such capacity they may act as hormone-releasing or inhibiting factors. Finally, endorphins appear to play a role in the behavioral concomitants of stress. In such capacity endorphins are suggested to function as modulators of neural systems that mediate the elaboration and expression of the reactive/affective components of stress. Speculations on the mode of interaction between endorphins and ACTH in the global response to stress are discussed.

Endogenous opioid ligands may mediate stress-induced changes in the affective properties of pain related behavior in rats.

Abstract The effect of a single or repeated daily sessions of immobilization stress on hot plate-induced paw lick and escape responses was studied in rats. Immobilization prior to testing resulted in increased latency to escape while having no effect on paw lick response. Naloxone pretreatment reversed the effect of immobilization on escape behavior. The data suggest that immobilization-induced changes in pain related behavior may be mediated by an opiate receptor ligand system. Furthermore, they suggest that this endogenous system may be mediating the affective but not the sensory properties of pain related behavior.

The pituitary gland mediates acute and chronic pain responsiveness in stressed and non-stressed rats.

Abstract The effect of hypophysectomy on the responsiveness of rats to acute (hot plate test) and chronic (formalin test) pain was studied. Hypophysectomy did not alter hot plate behavior but increased the duration of the paw withdrawal in the formalin test. Pre-exposure of rats to immobilization stress caused a transient yet significant increase in escape latencies from a hot plate as well as a significant decrease in the duration of paw withdrawal in the formalin test. Hypophysectomy blocked the effect of stress on these behavioral manifestations of acute and chronic pain. The effect of hypophysectomy was not reversed by adrenocorticotropin pretreatment. These results suggest that centrally acting pituitary hormones may have a requisite function in normal and adaptive pain control in mammals.

A Circadian Rhythm in the Expression of PERIOD2 Protein Reveals a Novel SCN-Controlled Oscillator in the Oval Nucleus of the Bed Nucleus of the Stria Terminalis

Circadian rhythms in mammals are regulated not only globally by the master clock in the suprachiasmatic nucleus (SCN), but also locally by widely distributed populations of clock cells in the brain and periphery that control tissue-specific rhythmic outputs. Here we show that the oval nucleus of the bed nucleus of the stria terminalis (BNST-OV) exhibits a robust circadian rhythm in expression of the Period2 (PER2) clock protein. PER2 expression is rhythmic in the BNST-OV in rats housed under a light/dark cycle or in constant darkness, in blind rats, and in mice, and is in perfect synchrony with the PER2 rhythm of the SCN. Constant light or bilateral SCN lesions abolish the rhythm of PER2 in the BNST-OV. Large abrupt shifts in the light schedule transiently uncouple the BNST-OV rhythm from that of the SCN. Re-entrainment of the PER2 rhythm is faster in the SCN than in the BNST-OV, and it is faster after a delay than an advance shift. Bilateral adrenalectomy blunts the PER2 rhythm in the BNST-OV. Thus, the BNST-OV contains circadian clock cells that normally oscillate in synchrony with the SCN, but these cells appear to require both input from the SCN and circulating glucocorticoids to maintain their circadian oscillation. Taken together with what is known about the functional organization of the connections of the BNST-OV with systems of the brain involved in stress and motivational processes, these findings place BNST-OV oscillators in a position to influence specific physiological and behavioral rhythms downstream from the SCN clock.

Corticotropin-releasing factor projections from limbic forebrain and paraventricular nucleus of the hypothalamus to the region of the ventral tegmental area

Corticotropin-releasing factor (CRF) is a peptide neurotransmitter with high numbers of cell bodies found in limbic regions of the rat brain including the oval nucleus of the bed nucleus of the stria terminalis (BNSTov) and central nucleus of the amygdala (CeA) as well as in the paraventricular nucleus of the hypothalamus (PVN). CRF systems are activated in response to acute stressors and mediate a wide variety of physiological and behavioral responses to acute stress including aversive responses and responses that support appetitive behaviors. CRF is released in the ventral tegmental area (VTA), the cell body region of the mesocorticolimbic dopaminergic neurons, in response to acute stress and plays a role in stress-activation of appetitive behavior [Wang B, Shaham Y, Zitzman D, Azari S, Wise RA, You ZB (2005) Cocaine experience establishes control of midbrain glutamate and dopamine by corticotropin-releasing factor: a role in stress-induced relapse to drug seeking. J Neurosci 25:5389-5396]. However, although it is known that the VTA region contains significant levels of CRF-immunoreactive fibers [Swanson LW, Sawchenko PE, Rivier J, Vale WW (1983) Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: an immunohistochemical study. Neuroendocrinology 36:165-186], the source of CRF input to the region has not been identified. We used infusions of a fluorescent retrograde tracer, fluorogold, into the VTA region, combined with fluorescent immunocytochemistry for CRF to identify sources of this input. Double-labeled cells were found in BNSTov, CeA and PVN. The percent of fluorogold-labeled cells in each region that were CRF-positive was 30.8, 28.0 and 16.7% respectively. These data point to diffusely distributed sources of CRF-containing fibers in the VTA.

Endogenous Dopamine Regulates the Rhythm of Expression of the Clock Protein PER2 in the Rat Dorsal Striatum via Daily Activation of D2 Dopamine Receptors

A role for dopamine (DA) in the regulation of clock genes in the mammalian brain is suggested by evidence that manipulations of DA receptors can alter the expression of some clock genes outside the suprachiasmatic nucleus (SCN), the master circadian clock. The role of endogenous DA in the regulation of clock gene expression is unknown. Here, we demonstrate a direct relationship between extracellular DA levels and the rhythm of expression of the clock protein PERIOD2 (PER2) in the dorsal striatum of the male Wistar rat. Specifically, we show that the peak of the daily rhythm of extracellular DA in the dorsal striatum precedes the peak of PER2 by ∼6 h and that depletion of striatal DA by 6-hydroxydopamine or α-methyl-para-tyrosine or blockade of D2 DA receptors by raclopride blunts the rhythm of striatal PER2. Furthermore, timed daily activation of D2 DA receptors, but not D1 DA receptors, restores and entrains the PER2 rhythm in the DA-depleted striatum. None of these manipulations had any effect on the PER2 rhythm in the SCN. Our findings are consistent with the idea that the rhythm of expression of PER2 in the dorsal striatum depends on daily dopaminergic activation of D2 DA receptors. These observations may have implications for circadian abnormalities seen in Parkinsons disease.

Glucocorticoid rhythms control the rhythm of expression of the clock protein, Period2, in oval nucleus of the bed nucleus of the stria terminalis and central nucleus of the amygdala in rats.

We investigated the involvement of the adrenal glucocorticoid, corticosterone, in the control of the rhythmic expression of the circadian clock protein, Period2, in forebrain nuclei known to be sensitive to glucocorticoids, stressors and drugs of abuse, the oval nucleus of the bed nucleus of the stria terminalis and the central nucleus of the amygdala. We found previously that the daily rhythm of Period2 in these nuclei is uniquely dependent on the integrity of the adrenal glands (Amir S, Lamont EW, Robinson B, Stewart J (2004) A circadian rhythm in the expression of PERIOD2 protein reveals a novel SCN-controlled oscillator in the oval nucleus of the bed nucleus of the stria terminalis. J Neurosci 24:781-790; Lamont EW, Robinson B, Stewart J, Amir S (2005) The central and basolateral nuclei of the amygdala exhibit opposite diurnal rhythms of expression of the clock protein Period2. Proc Natl Acad Sci U S A 102:4180-4184). We now show that, in rats, in the absence of the adrenals, corticosterone replacement via the drinking water, which is associated with daily fluctuations in corticosterone levels, restores the rhythm of Period2 in the oval nucleus of the bed nucleus of the stria terminalis and central nucleus of the amygdala. Corticosterone replacement via constant-release pellets has no effect. These results underscore the importance of circadian glucocorticoid signaling in Period2 rhythms in the oval nucleus of the bed nucleus of the stria terminalis and central nucleus of the amygdala and suggest a novel mechanism whereby stressors, drugs of abuse, and other abnormal states that affect the patterns of circulating glucocorticoids can alter the functional output of these nuclei.

Differential regulation of the expression of period2 protein in the limbic forebrain and dorsomedial hypothalamus by daily limited access to highly palatable food in food-deprived and free-fed rats

Circadian clock genes are rhythmically expressed in many areas of the brain and body and are thought to underlie most endogenous circadian behaviors and physiological processes. Daily rhythms of clock gene expression throughout the brain and body are normally coordinated by the suprachiasmatic nucleus (SCN), but they are also strongly influenced by daily temporal restrictions of food availability. Here, we studied the effects of a daily restricted presentation of highly palatable complete meal replacement, chocolate Ensure Plus (Ensure) in food-deprived (restricted feeding, RF) and free-fed (restricted treat, RT) rats, on the expression of the clock protein, Period2 (PER2) in regions of the brain involved in motivational and emotional regulation; these include the oval nucleus of the bed nucleus of the stria terminalis (BNSTov), the central nucleus of the amygdala (CEA), the basolateral amygdala (BLA), the dentate gyrus (DG) and the dorsomedial hypothalamus (DMH). RF and RT rats consumed similar amounts of Ensure, but changes in the pattern of PER2 expression were seen only in the RF condition, suggesting that changes in PER2 expression in these regions are triggered by the daily alleviation of a negative metabolic state associated with RF and are independent of the positive incentive properties of the consumed substance, per se. In contrast, the expression of the immediate early gene, Fos, was increased in these regions by both RF and RT schedules, showing that signals concerning the incentive value of the consumed food reach these regions. No changes in either PER2 or Fos expression were observed in the SCN of RF or RT rats. These findings demonstrate that mechanisms leading to changes in the expression of PER2 and those affecting the induction of Fos under RF and RT are, at least in part, dissociable.

Food‐entrainable circadian oscillators in the brain

Circadian rhythms in mammalian behaviour and physiology rely on daily oscillations in the expression of canonical clock genes. Circadian rhythms in clock gene expression are observed in the master circadian clock, the suprachiasmatic nucleus but are also observed in many other brain regions that have diverse roles, including influences on motivational and emotional state, learning, hormone release and feeding. Increasingly, important links between circadian rhythms and metabolism are being uncovered. In particular, restricted feeding (RF) schedules which limit food availability to a single meal each day lead to the induction and entrainment of circadian rhythms in food‐anticipatory activities in rodents. Food‐anticipatory activities include increases in core body temperature, activity and hormone release in the hours leading up to the predictable mealtime. Crucially, RF schedules and the accompanying food‐anticipatory activities are also associated with shifts in the daily oscillation of clock gene expression in diverse brain areas involved in feeding, energy balance, learning and memory, and motivation. Moreover, lesions of specific brain nuclei can affect the way rats will respond to RF, but have generally failed to eliminate all food‐anticipatory activities. As a consequence, it is likely that a distributed neural system underlies the generation and regulation of food‐anticipatory activities under RF. Thus, in the future, we would suggest that a more comprehensive approach should be taken, one that investigates the interactions between multiple circadian oscillators in the brain and body, and starts to report on potential neural systems rather than individual and discrete brain areas.

Circadian modulation of Fos responses to odor of the red fox, a rodent predator, in the rat olfactory system

We have previously shown that neuronal responses to a biologically neutral odor, cedar wood oil, in the olfactory system are greater in the subjective night compared to subjective day. In the present study, we confirm these results and extend them to a biologically relevant odor, the urine of the red fox, a rodent predator. Fos induced by exposure of rats to fox urine or a neutral odor, mineral oil, was markedly enhanced during the subjective night compared to subjective day in the main olfactory bulb, primary olfactory cortex, and other structures related to olfaction. These results show that neuronal responses to an ethologically relevant odor follow a circadian rhythm similar to biologically neutral odors. Fos responses induced by fox urine were observed to be of greater magnitude than a neutral odor in brain areas involved in fear responses, suggesting that fox urine activates fear circuitry.