F Santoru

Morphine withdrawal produces ERK-dependent and ERK-independent epigenetic marks in neurons of the nucleus accumbens and lateral septum

Epigenetic changes such as covalent modifications of histone proteins represent complex molecular signatures that provide a cellular memory of previously experienced stimuli without irreversible changes of the genetic code. In this study we show that new gene expression induced in vivo by morphine withdrawal occurs with concomitant epigenetic modifications in brain regions critically involved in drug-dependent behaviors. We found that naloxone-precipitated withdrawal, but not chronic morphine administration, caused a strong induction of phospho-histone H3 immunoreactivity in the nucleus accumbens (NAc) shell/core and in the lateral septum (LS), a change that was accompanied by augmented H3 acetylation (lys14) in neurons of the NAc shell. Morphine withdrawal induced the phosphorylation of the epigenetic factor methyl-CpG-binding protein 2 (MeCP2) in Ser421 both in the LS and the NAc shell. These epigenetic changes were accompanied by the activation of members of the ERK pathway as well as increased expression of the immediate early genes (IEG) c-fos and activity-regulated cytoskeleton-associated protein (Arc/Arg3.1). Using a pharmacological approach, we found that H3 phosphorylation and IEG expression were partially dependent on ERK activation, while MeCP2 phosphorylation was fully ERK-independent. These findings provide new important information on the role of the ERK pathway in the regulation of epigenetic marks and gene expression that may concur to regulate in vivo the cellular changes underlying the onset of the opioid withdrawal syndrome.

Neonatal exposure to estradiol in rats influences neuroactive steroid concentrations, GABAA receptor expression, and behavioral sensitivity to anxiolytic drugs.

J. Neurochem. (2010) 113, 1285–1295.

Long-term administration with levonorgestrel decreases allopregnanolone levels and alters GABAA receptor subunit expression and anxiety-like behavior

Fluctuations in the concentrations of the neuroactive steroid allopregnanolone are thought to influence γ-amino-butyric acid type A (GABA(A)) receptor gene expression and function. Long-term treatment with ethinyl estradiol (EE) plus levonorgestrel (LNG), two of the most widely used steroids in the hormonal contraceptive pill, decreases allopregnanolone levels in rat cerebral cortex and plasma, alters GABA(A) receptor expression and induces anxiety-like behavior. We evaluated which component of the hormonal contraceptive pill is responsible for the aforementioned changes. Female rats were injected subcutaneously (s.c.) with EE (0.030 mg) or LNG (0.125 mg) once a day for 4 weeks. Compared to the respective vehicle-treated control groups, EE decreased cerebral cortical levels of allopregnanolone, progesterone and pregnenolone by 76, 72 and 33%, respectively and hippocampal levels by 52, 56 and 50%, respectively. Likewise, LNG decreased cerebral cortical levels of allopregnanolone, progesterone and pregnenolone by 75, 68 and 33%, respectively, and hippocampal levels by 55, 65 and 60%, respectively. Administration of LNG, but not EE, increased the abundance of the γ2 subunit peptide in cerebral cortex and hippocampus by 38 and 59%, respectively. Further, LNG, but not EE, decreased the time spent and the number of entries into the open arms of the elevated plus maze by 56 and 43%, respectively, an index of anxiety-like behavior. These results suggest that alterations in GABA(A) receptor subunit expression and anxiety-like behavior induced by long-term treatment with combined EE/LNG appear to be caused by LNG. Given that both EE and LNG decrease allopregnanolone levels in a similar manner, these results further suggest that changes in allopregnanolone levels are not associated with GABA(A) receptor expression.

Failure of Acute Ethanol Administration to Alter Cerebrocortical and Hippocampal Allopregnanolone Levels in C57BL/6J and DBA/2J Mice

BACKGROUND Ethanol (EtOH) administration increases brain allopregnanolone levels in rats, and this increase contributes to sensitivity to EtOHs behavioral effects. However, EtOHs effects on allopregnanolone may differ across species. We investigated the effects of acute EtOH administration on allopregnanolone, progesterone, and corticosterone levels in cerebral cortex and hippocampus of C57BL/6J and DBA/2J mice, 2 inbred strains with different alcohol sensitivity. METHODS Naïve male C57BL/6J and DBA/2J mice received EtOH (1, 2, 3, or 4 g/kg, intraperitoneally [i.p.]) or saline and were euthanized 1 hour later. For the time-course study, mice received EtOH (2 g/kg, i.p.) and were euthanized 15, 30, 60, and 120 minutes later. Steroids were measured by radioimmunoassay. RESULTS Acute EtOH administration did not alter cerebrocortical and hippocampal levels of allopregnanolone and progesterone in these strains at any of the doses and time points examined. Acute EtOH dose-dependently increased cerebrocortical corticosterone levels by 319, 347, and 459% in C57BL/6J mice at the doses of 2, 3, and 4 g/kg, and by 371, 507, 533, and 692% in DBA/2J mice at the doses of 1, 2, 3, and 4 g/kg, respectively. Similar changes were observed in the hippocampus. EtOHs effects on cerebrocortical corticosterone levels were also time dependent in both strains. Moreover, acute EtOH administration time-dependently increased plasma levels of progesterone and corticosterone. Finally, morphine administration increased cerebrocortical allopregnanolone levels in C57BL/6J (+77, +93, and +88% at 5, 10, and 30 mg/kg, respectively) and DBA/2J mice (+81% at 5 mg/kg), suggesting that the impairment in brain neurosteroidogenesis may be specific to EtOH. CONCLUSIONS These results underline important species differences on EtOH-induced brain neurosteroidogenesis. Acute EtOH increases brain and plasma corticosterone levels but does not alter cerebrocortical and hippocampal concentrations of allopregnanolone and progesterone in naïve C57BL/6J and DBA/2J mice.

Neonatal exposure to estradiol decreases hypothalamic allopregnanolone concentrations and alters agonistic and sexual but not affective behavior in adult female rats

Exposure of developing female rats to estradiol during the perinatal period induced long-lasting dysregulation of gonadal axis and decreased cerebrocortical and plasma concentrations of allopregnanolone. We have now examined the effects of neonatal estradiol administration in female rats on hypothalamic allopregnanolone concentrations and on exploratory, affective, agonistic and sexual behaviors as well as social learning. A single administration of β-estradiol 3-benzoate (EB, 10μg) on the day of birth resulted in a delay of vaginal opening, acyclicity and ovarian failure. These alterations were associated with a significant decrease in the concentrations of allopregnanolone in the hypothalamus at 21 and 60days, but not at 7days, after birth. Neonatal administration of EB also increased agonistic behaviors in adult rats, such as dominant behaviors and following of an ovariectomized intruder, while living attacks unaffected. EB-treated rats showed also an increase in anogenital investigation, associated with a drastic reduction in spontaneous and induced female sexual behaviors (receptivity and proceptivity). In contrast, neonatal administration of EB did not affect locomotor activity, anxiety- and mood-related behaviors, the social transmission of flavor preferences, and seizures sensitivity. These effects of estradiol suggest that it plays a major role in regulation of both the abundance of allopregnanolone and the expression of agonistic and sexual behaviors, while failing to influence affective behaviors and social learning. Thus, the pronounced and persistent decrease in hypothalamic allopregnanolone concentration may be related to the manifestation of agonistic and sexual behaviors.

Neonatal estradiol exposure to female rats changes GABAA receptor expression and function, and spatial learning during adulthood.

&NA; Exposure of female rats to estradiol during the perinatal period has profound effects on GABAergic neurotransmission that are crucial to establish sexually dimorphic brain characteristics. We previously showed that neonatal &bgr;‐estradiol 3‐benzoate (EB) treatment decreases brain concentrations of the neurosteroid allopregnanolone, a potent positive modulator of extrasynaptic GABAA receptors (GABAAR). We thus evaluated whether neonatal EB treatment affects GABAAR expression and function in the hippocampus of adult female rats. Neonatal EB administration increased the expression of extrasynaptic &agr;4/&dgr; subunit‐containing GABAARs and the modulatory action of THIP on tonic currents mediated by these receptors. The same treatment decreased the expression of synaptic &agr;1/&agr;4/&ggr;2 subunit‐containing receptors, as well as phasic currents. These effects of neonatal EB treatment are not related to ambient allopregnanolone concentrations per se, given that vehicle‐treated rats in diestrus, which have opposite neurosteroid levels than EB‐treated rats, show similar changes in GABAARs. Rather, these changes may represent a compensatory mechanism to counteract the long‐term reduction in allopregnanolone concentrations, induced by neonatal EB. Given that both &agr;4/&dgr; receptors and allopregnanolone are involved in memory consolidation, we evaluated whether neonatal EB treatment alters performance in the Morris water maze test during adulthood. Neonatal EB treatment decreased the latency and the cumulative search error to reach the platform, as well as thigmotaxis, suggesting improved learning, and also enhanced memory performance during the probe trial. These enduring changes in GABAAR plasticity may be relevant for the regulation of neuronal excitability in the hippocampus and for the etiology of psychiatric disorders that originate in development and show sex differences. HighlightsNeonatal estradiol (EB) increases extrasynaptic &agr;4/&dgr; GABAARs in adult hippocampus.Neonatal EB decreases synaptic &agr;1/&agr;4/&ggr;2 GABAAR subunits in adult rat hippocampus.Neonatal EB alters GABAergic tonic and phasic currents in adult rat hippocampus.These changes in GABAARs are independent of ambient allopregnanolone levels.Neonatal EB improves spatial learning and memory retrieval in adult rats.