Maria Luisa Laorden

Effects of morphine and morphine withdrawal on brainstem neurons innervating hypothalamic nuclei that control thepituitary-adrenocortical axis in rats

Different data support a role for brainstem noradrenergic inputs to the hypothalamic paraventricular nucleus (PVN) in the control of hypothalamus – pituitary – adrenocortical (HPA) axis. However, little is known regarding the functional adaptive changes of noradrenergic afferent innervating the PVN and supraoptic nucleus (SON) during chronic opioid exposure and upon morphine withdrawal. Here we have studied the expression of Fos after administration of morphine and during morphine withdrawal in the rat hypothalamic PVN and SON. Fos production was also studied in brainstem regions that innervate hypothalamic nuclei: the nucleus of solitary tract (NTS – A2) and the ventrolateral medulla (VLM – A1) and combined with immunostaining for tyrosine hydroxylase (TH) for immunohistochemical identification of active neurons during morphine withdrawal. Male rats were implanted with s.c. placebo or morphine (tolerant/dependent) pellets for 7 days. On day 8 rats received an injection of saline i.p., morphine i.p., saline s.c. or naloxone s.c. Acute morphine administration produced an increase in Fos expression at hypothalamic nuclei and in the brainstem regions, and tolerance developed towards this effect. Precipitated morphine withdrawal induced marked Fos immunoreactivity within the PVN and SON. Concomitantly, numerous neurons in the brainstem were stimulated by morphine withdrawal. Moreover, catecholaminergic‐positive neurons in the brainstem showed a significant increase in Fos expression in response to morphine withdrawal. These findings demonstrate that chronic activation of opioid receptors results in altered patterns of immediate‐early genes (IEG) expression in the PVN and SON, which occurs concurrently with an increased activity of their inputs from the brainstem.

Morphine regulates Argonaute 2 and TH expression and activity but not miR‐133b in midbrain dopaminergic neurons

Epigenetic changes such as microRNAs (miRs)/Ago2‐induced gene silencing represent complex molecular signature that regulate cellular plasticity. Recent studies showed involvement of miRs and Ago2 in drug addiction. In this study, we show that changes in gene expression induced by morphine and morphine withdrawal occur with concomitant epigenetic modifications in the mesolimbic dopaminergic (DA) pathway [ventral tegmental area (VTA)/nucleus accumbens (NAc) shell], which is critically involved in drug‐induced dependence. We found that acute or chronic morphine administration as well as morphine withdrawal did not modify miR‐133b messenger RNA (mRNA) expression in the VTA, whereas Ago2 protein levels were decreased and increased in morphine‐dependent rats and after morphine withdrawal, respectively. These changes were paralleled with enhanced and decreased NAc tyrosine hydroxylase (TH) protein (an early DA marker) in morphine‐dependent rats and after withdrawal, respectively. We also observed changes in TH mRNA expression in the VTA that could be related to Ago2‐induced translational repression of TH mRNA during morphine withdrawal. However, the VTA number of TH‐positive neurons suffered no alterations after the different treatment. Acute morphine administration produced a marked increase in TH activity and DA turnover in the NAc (shell). In contrast, precipitated morphine withdrawal decreased TH activation and did not change DA turnover. These findings provide new information into the possible correlation between Ago2/miRs complex regulation and DA neurons plasticity during opiate addiction.

Effects of U‐50,488H and U‐50,488H withdrawal on c‐fos expression in the rat paraventricular nucleus. Correlation with c‐fos in brainstem catecholaminergic neurons

In the present work, we have studied the expression of Fos during acute and chronic administration of the κ‐opioid receptor agonist U‐50,488H and after U‐50,88H withdrawal in the rat hypothalamic paraventricular nucleus (PVN). Fos production was also studied in brainstem regions that innervate the PVN: the A2 cell group of the nucleus of solitary tract (NTS‐A2) and the A1 cell group of the ventrolateral medulla (VLM‐A1), combined with immunostaining for tyrosine hydroxylase (TH) for immunohistochemical identification of active neurons after acute U‐50,488H administration. For acute experiments, male rats were treated with saline i.p. for 4 days. On day 5, rats were given saline or U‐50,488H (15 mg kg−1, i.p.). Other groups of rats were rendered tolerant/dependent on U‐50,488H by injecting the drug twice daily (15 mg kg−1, i.p.) for 4 days. Control animals received saline i.p. on the same time schedule. On day 5, rats were treated with vehicle i.p., with U‐50,488H (15 mg kg−1) or with the selective κ opioid‐receptor antagonist nor‐binaltorphimine (Nor‐BNI, 5 mg kg−1, i.p.). Using immunohistochemical staining of Fos, present results indicate that acute administration of U‐50,488H produced an increase in Fos expression in the PVN and in the noradrenergic A1 and A2 cell groups. Moreover, when double‐label immunohistochemistry was used to identify Fos and catecholaminergic‐positive neurons in the brainstem, it was found that catecholaminergic‐positive neurons in the NTS and VLM showed a significant increase in Fos expression in response to acute U‐50,488H injection. Chronic application of U‐50,488H leads to the development of tolerance towards their effects on Fos expression in the PVN as well as in the NTS and VLM. However, administration of Nor‐BNI to U‐50,488H‐dependent rats did not induce any changes in Fos immunoreactivity in the PVN or in the brainstem. These findings demonstrate that acute activation of κ‐opioid receptors results in different altered patterns of immediate‐early gene expression in the PVN, which occurs concurrently with an increased activity of their inputs from the brainstem. Interestingly in contrast to morphine withdrawal, present results demonstrate that rats withdrawn from U‐50,488H did show no changes in Fos‐immunoreactivity in the PVN, NTS or VLM, indicating the absence of dependence on the κ‐agonist under the present experimental conditions.

Sex Differences between CRF1 Receptor Deficient Mice following Naloxone-Precipitated Morphine Withdrawal in a Conditioned Place Aversion Paradigm: Implication of HPA Axis

Background Extinction period of positive affective memory of drug taking and negative affective memory of drug withdrawal, as well as the different response of men and women might be important for the clinical treatment of drug addiction. We investigate the role of corticotropin releasing factor receptor type one (CRF1R) and the different response of male and female mice in the expression and extinction of the aversive memory. Methodology/Principal Finding We used genetically engineered male and female mice lacking functional CRF1R. The animals were rendered dependent on morphine by intraperitoneally injection of increasing doses of morphine (10–60 mg/kg). Negative state associated with naloxone (1 mg/kg s.c.)-precipitated morphine withdrawal was examined by using conditioned place aversion (CPA) paradigm. No sex differences for CPA expression were found in wild-type (n = 29) or CRF1R knockout (KO) mice (n = 29). However, CRF1R KO mice presented less aversion score than wild-type mice, suggesting that CRF1R KO mice were less responsive than wild-type to continuous associations between drug administration and environmental stimuli. In addition, CPA extinction was delayed in wild-type and CRF1R KO male mice compared with females of both genotypes. The genetic disruption of the CRF1R pathway decreased the period of extinction in males and females suggesting that CRF/CRF1R is implicated in the duration of aversive memory. Our results also showed that the increase in adrenocorticotropic hormone (ACTH) levels observed in wild-type (n = 11) mice after CPA expression, were attenuated in CRF1R KO mice (n = 10). In addition, ACTH returned to the baseline levels in males and females once CPA extinction was finished. Conclusion/Significance These results suggest that, at least, CPA expression is partially due to an increase in plasma ACTH levels, through activation of CRF1R, which can return when CPA extinction is finished.

Regulation of Pleiotrophin, Midkine, Receptor Protein Tyrosine Phosphatase β/ζ, and Their Intracellular Signaling Cascades in the Nucleus Accumbens During Opiate Administration.

Background: Most classes of addictive substances alter the function and structural plasticity of the brain reward circuitry. Midkine (MK) and pleiotrophin (PTN) are growth/differentiation cytokines which, similarly to neurotrophins, play an important role in repair, neurite outgrowth, and cell differentiation. PTN or MK signaling through receptor protein tyrosine phosphatase β/ζ (RPTPβ/ζ), leads to the activation of extracellular signal-regulated kinases and thymoma viral proto-oncogene. This activation induces morphological changes and modulates addictive behaviors. Besides, there is increasing evidence that during the development of drug addiction, astrocytes contribute to the synaptic plasticity by synthesizing and releasing substances such as cytokines. Methods: In the present work we studied the effect of acute morphine administration, chronic morphine administration, and morphine withdrawal on PTN, MK, and RPTPβ/ζ expression and on their signaling pathways in the nucleus accumbens. Results: Present results indicated that PTN, MK, and RPTPβ/ζ levels increased after acute morphine injection, returned to basal levels during chronic opioid treatment, and were up-regulated again during morphine withdrawal. We also observed an activation of astrocytes after acute morphine injection and during opiate dependence and withdrawal. In addition, immunofluorescence analysis revealed that PTN, but not MK, was overexpressed in astrocytes and that dopaminoceptive neurons expressed RPTPβ/ζ. Conclusions: All these observations suggest that the neurotrophic and behavioral adaptations that occur during opiate addiction could be, at least partly, mediated by cytokines.