Differential Changes in Expression of Stress- and Metabolic-related Neuropeptides in the Rat Hypothalamus during Morphine Dependence and Withdrawal

Chronic morphine treatment and naloxone precipitated morphine withdrawal activates stress-related brain circuit and results in significant changes in food intake, body weight gain and energy metabolism. The present study aimed to reveal hypothalamic mechanisms underlying these effects. Adult male rats were made dependent on morphine by subcutaneous implantation of constant release drug pellets. Pair feeding revealed significantly smaller weight loss of morphine treated rats compared to placebo implanted animals whose food consumption was limited to that eaten by morphine implanted pairs. These results suggest reduced energy expenditure of morphine-treated animals. Chronic morphine exposure or pair feeding did not significantly affect hypothalamic expression of selected stress- and metabolic related neuropeptides - corticotropin-releasing hormone (CRH), urocortin 2 (UCN2) and proopiomelanocortin (POMC) compared to placebo implanted and pair fed animals. Naloxone precipitated morphine withdrawal resulted in a dramatic weight loss starting as early as 15-30 min after naloxone injection and increased adrenocorticotrophic hormone, prolactin and corticosterone plasma levels in morphine dependent rats. Using real-time quantitative PCR to monitor the time course of relative expression of neuropeptide mRNAs in the hypothalamus we found elevated CRH and UCN2 mRNA and dramatically reduced POMC expression. Neuropeptide Y (NPY) and arginine vasopressin (AVP) mRNA levels were transiently increased during opiate withdrawal. These data highlight that morphine withdrawal differentially affects expression of stress- and metabolic-related neuropeptides in the rat hypothalamus, while relative mRNA levels of these neuropeptides remain unchanged either in rats chronically treated with morphine or in their pair-fed controls

A stressz rendszer szerepe az ópiát függőség kialakulásában: idegi, sejtszintű és molekuláris mechanizmusok = Involvement of brain stress system in opiate addiction: neuronal substrates, cellular and molecular mechanisms

1. A morfium függőség és megvonás aktiválja a neuroendokrin és az agyi stresszrendszert. 2. A hipotalamusz paravntrikuláris magjában illetve az ""extended"" amigdalában expresszálódó kortikotropin-releasing hormon (CRH) különböző módon szabályozza a morfium addikciót és megvonást kísérő hormonális, élettani és magatartási (érzelmi) reakciókat. 3. Sejtszinten a cAMP-reponse element binding protein (CREB) és a hozzá csatlakozó ko-regulátor fehérjék, mint pl a foszforilált TORC fontos szerepet játszanak a drogmegvonás kapcsán aktiválódó CRH gén szabályozásában mind a hipotalamuszban, mind az ""extended"" amigdalában. 4. A hipotalamikus és extrahipotalamikus területeket beidegző felszálló noradrenerg és orexinerg pályák különböző mechanizmussal vesznek részt a drogfüggőséggel és a drogmegvonással járó megvonási tünetek kialakításában. 5. A morfium függőséggel és a naloxonnal történő morfium megvonás okozta anyagcsere változások a hipotalamuszban eltérő módon befolyásolják a stresszel és a metabolikus szabályozássaél kapcsolatos neuropaptid gének expresszióját. | 1. Morphine dependence and naloxone-precipitated morphine withdrawal results in activation of the neuroendocrine and brain stress systems in the rat brain. 2. Corticotropin-releasing hormone expressed in the hypophyseotropic cells of the hypothalamus as well as in the extended amygdala plays a differential role in the development of hormonal, physiological, emotional and behavioral changes in addicted animals. 3. cAMP-response element binding protein (CREB) and its co-activators (such as pTORC) play a critical role in activation of corticotropin-releasing hormone gene expression both in the hypothalamus and in the extended amygdala. 4. Ascending noradrenergic and orexinergic pathways innervating hypothalamic and extrahypothalamic sites differentially regulate the development of hormonal, somatic and psychological symptoms of morphine withdrawal. 5. Metabolic changes associated by drug dependence and withdrawal result in differential changes of metabolic and stress-related neuropeptides in the hypothalamus

Hypothalamic orexin--a neurons are involved in the response of the brain stress system to morphine withdrawal.

Both the hypothalamus-pituitary-adrenal (HPA) axis and the extrahypothalamic brain stress system are key elements of the neural circuitry that regulates the negative states during abstinence from chronic drug exposure. Orexins have recently been hypothesized to modulate the extended amygdala and to contribute to the negative emotional state associated with dependence. This study examined the impact of chronic morphine and withdrawal on the lateral hypothalamic (LH) orexin A (OXA) gene expression and activity as well as OXA involvement in the brain stress response to morphine abstinence. Male Wistar rats received chronic morphine followed by naloxone to precipitate withdrawal. The selective OX1R antagonist SB334867 was used to examine whether orexins' activity is related to somatic symptoms of opiate withdrawal and alterations in HPA axis and extended amygdala in rats dependent on morphine. OXA mRNA was induced in the hypothalamus during morphine withdrawal, which was accompanied by activation of OXA neurons in the LH. Importantly, SB334867 attenuated the somatic symptoms of withdrawal, and reduced morphine withdrawal-induced c-Fos expression in the nucleus accumbens (NAc) shell, bed nucleus of stria terminalis, central amygdala and hypothalamic paraventricular nucleus, but did not modify the HPA axis activity. These results highlight a critical role of OXA signalling, via OX1R, in activation of brain stress system to morphine withdrawal and suggest that all orexinergic subpopulations in the lateral hypothalamic area contribute in this response

Plasma hormone levels and blood glucose concentration 7 days after morphine or placebo implantation and 2 hours after saline or naloxone injection.

+<p>p<0,05 placebo+saline vs. placebo+naloxone; *p<0,05 morphine+saline vs. morphine+naloxone group; n = 6/group.</p

Effect of chronic morphine treatment and naloxone precipitated morphine withdrawal on neuropeptide expression in the hypothalamus.

<p>Mean (±SEM) values of relative expression values of selected hypothalamic neuropeptide mRNAs in placebo or morphine treated rats two hours after saline or naloxone injection. Neuropeptide mRNA levels were normalised to GAPDH levels and expressed as fold change compared to the levels of placebo implanted saline groups set to 1. n = 6/group, *p<0.0.1 compared to saline injected respective controls.</p

Mean±SEM relative expression levels of selected hypothalamic neuropeptides in placebo or morphine-treated and pair fed animals 2 or 7 days after pellet implantation.

*<p><i>p</i><0,01 placebo vs. pair fed; ^#<i>p</i><0,01 morphine vs. pair fed; ⁺<i>p</i><0,001 placebo vs. morphine; n = 7/group.</p

Schematic overview of the pair feeding and time course experiments.

<p>Baseline food intake (FI) and body weight (BW) data were obtained on days −2 and −1. On day 0 rats were subcutaneously implanted with placebo or morphine pellets. Based on the food consumption data of morphine implanted rats, a group of placebo implanted, pair fed rats was added on day 1. These rats received the same amount of food daily that was consumed by their morphine-implanted pairs on the previous day. Some morphine and placebo treated rats were decapitated on day 2 along their placebo-implanted, pair fed partners on day 3. On day 7, placebo and morphine-implanted groups were further divided into two groups half of the rats from each group received subcutaneous injection of saline or naloxone and sacrificed at different time points thereafter. Animals from pair fed group were decapitated on day 8 (7^th day after pellet implantation).</p

List of primer pairs used in real-time quantitative PCR reactions.

<p>List of primer pairs used in real-time quantitative PCR reactions.</p

Effects of chronic morphine treatment and pair feeding on metabolic parameters of male rats.

<p>Daily changes of food intake in placebo and morphine implanted animals. Pair fed rats were placebo implanted and received the same amount of food that was consumed by the morphine implanted pairs. n = 12, ⁺<i>p</i><0.05, ⁺⁺<i>p</i><0.01 morphine vs. placebo-implanted group (Student’s t-test) A. Mean (±SEM) values of daily changes in body weight of morphine implanted and placebo implanted and pair fed rats. Note that pair feeding of placebo implanted rats resulted in significantly more body weight loss compared to morphine implanted animals, n = 12, *<i>p</i><0.05 placebo implanted vs. pair fed group (Student’s t-test).</p