Morphine-withdrawal aversive memories and their extinction modulate H4K5 acetylation and Brd4 activation in the rat hippocampus and basolateral amygdala.

Chromatin modification is a crucial mechanism in several important phenomena in the brain, including drug addiction. Persistence of drug craving and risk of relapse could be attributed to drug-induced epigenetic mechanisms that seem to be candidates explaining long-lasting drug-induced behaviour and molecular alterations. Histone acetylation has been proposed to regulate drug-seeking behaviours and the extinction of rewarding memory of drug taking. In this work, we studied the epigenetic regulation during conditioned place aversion and after extinction of aversive memory of opiate withdrawal. Through immunofluorescence assays, we assessed some epigenetic marks (H4K5ac and p-Brd4) in crucial areas related to memory retrieval -basolateral amygdala (BLA) and hippocampus-. Additionally, to test the degree of transcriptional activation, we evaluated the immediate early genes (IEGs) response (Arc, Bdnf, Creb, Egr-1, Fos and Nfkb) and Smarcc1 (chromatin remodeler) through RT-qPCR in these nuclei. Our results showed increased p-Brd4 and H4K5ac levels during aversive memory retrieval, suggesting a more open chromatin state. However, transcriptional activation of these IEGs was not found, therefore suggesting that other secondary response may already be happening. Additionally, Smarcc1 levels were reduced due to morphine chronic administration in BLA and dentate gyrus. The activation markers returned to control levels after the retrieval of aversive memories, revealing a more repressed chromatin state. Taken together, our results show a major role of the tandem H4K5ac/p-Brd4 during the retrieval of aversive memories. These results might be useful to elucidate new molecular targets to improve and develop pharmacological treatments to address addiction and to avoid drug relapse.

Induction of FosB/DeltaFosB in the brain stress system-related structures during morphine dependence and withdrawal.

The transcription factor DeltaFosB is induced in the nucleus accumbens (NAc) by drugs of abuse. This study was designed to evaluate the possible modifications in FosB/DeltaFosB expression in both hypothalamic and extrahypothalamic brain stress system during morphine dependence and withdrawal. Rats were made dependent on morphine and, on day 8, were injected with saline or naloxone. Using immunohistochemistry and western blot, the expression of FosB/DeltaFosB, tyrosine hydroxylase (TH), corticotropin-releasing factor (CRF) and pro-dynorphin (DYN) was measured in different nuclei from the brain stress system in morphine-dependent rats and after morphine withdrawal. Additionally, we studied the expression of FosB/DeltaFosB in CRF-, TH- and DYN-positive neurons. FosB/DeltaFosB was induced after chronic morphine administration in the parvocellular part of the hypothalamic paraventricular nucleus (PVN), NAc-shell, bed nucleus of the stria terminalis, central amygdala and A(2) noradrenergic part of the nucleus tractus solitarius (NTS-A(2)). Morphine dependence and withdrawal evoked an increase in FosB/DeltaFosB-TH and FosB/DeltaFosB-CRF double labelling in NTS-A(2) and PVN, respectively, besides an increase in TH levels in NTS-A(2) and CRF expression in PVN. These data indicate that neuroadaptation to addictive substances, observed as accumulation of FosB/DeltaFosB, is not limited to the reward circuits but may also manifest in other brain regions, such as the brain stress system, which have been proposed to be directly related to addiction.

Phosphodiesterase 4 inhibitors, rolipram and diazepam block the adaptive changes observed during morphine withdrawal in the heart.

In this study, we investigated whether morphine dependence was inhibited by phosphodiesterase (PDE) 4 inhibitors rolipram and diazepam, since a role for the cyclic AMP systems in the development of morphine dependence was reported. Dependence of morphine was induced by a 7-day s.c. implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by an injection of naloxone. In order to determine the effect of rolipram or diazepam the animals were injected with these drugs for seven days and 30 min before the administration of naloxone. When opioid withdrawal was precipitated, enhancement of noradrenaline (NA) turnover in the heart was observed 30 min after naloxone administration. Moreover, morphine withdrawal induces Fos expression, increase in cyclic AMP and cyclic GMP levels. Co-administration of rolipram or diazepam with morphine during the pre-treatment period significantly reduces the signs of withdrawal symptoms, the enhancement of NA turnover, the increase in cyclic AMP and the Fos expression. However, these inhibitors did not modify the levels of cyclic GMP. These findings demonstrated that co-administration of rolipram or diazepam with morphine abolish the development of morphine dependence and suggest that these compounds prevent the up-regulation of the cyclic AMP pathway and the associated increase in cyclic AMP level after naloxone administration.

Glial activation and midkine and pleiotrophin transcription in the ventral tegmental area are modulated by morphine administration.

Opiates cause persistent restructuring in the mesolimbic reward system. Although a possible role for midkine and pleiotrophin cytokines in the field of synaptic plasticity has been proposed, it has not been assessed whether morphine administration regulates astrogliosis and midkine and pleiotrophin transcription. We observed that single morphine injection and chronic morphine increased glial fibrillary acidic protein expression in the ventral tegmental area (VTA). Interestingly, single morphine injection and chronic morphine increased VTA midkine and pleiotrophin mRNA expression. Given these results, we hypothesize a role for these cytokines in mediating, at least in part, acute neuroprotective effects and chronic neurotrophic adaptations that contribute to drug dependence.