Paola Morales

Ethanol as a Prodrug: Brain Metabolism of Ethanol Mediates its Reinforcing effects

BACKGROUND  While the molecular entity responsible for the rewarding effects of virtually all drugs of abuse is known, that for ethanol remains uncertain. Some lines of evidence suggest that the rewarding effects of alcohol are mediated not by ethanol per se but by acetaldehyde generated by catalase in the brain. However, the lack of specific inhibitors of catalase has not allowed strong conclusions to be drawn about its role on the rewarding properties of ethanol. The present studies determined the effect on voluntary alcohol consumption of two gene vectors, one designed to inhibit catalase synthesis and one designed to synthesize alcohol dehydrogenase (ADH), to respectively inhibit or increase brain acetaldehyde synthesis. METHODS  The lentiviral vectors, which incorporate the genes they carry into the cell genome, were (i) one encoding a shRNA anticatalase synthesis and (ii) one encoding alcohol dehydrogenase (rADH1). These were stereotaxically microinjected into the brain ventral tegmental area (VTA) of Wistar-derived rats bred for generations for their high alcohol preference (UChB), which were allowed access to an ethanol solution and water. RESULTS  Microinjection into the VTA of the lentiviral vector encoding the anticatalase shRNA virtually abolished (-94% p < 0.001) the voluntary consumption of alcohol by the rats. Conversely, injection into the VTA of the lentiviral vector coding for ADH greatly stimulated (2 to 3 fold p < 0.001) their voluntary ethanol consumption. CONCLUSIONS The study strongly suggests that to generate reward and reinforcement, ethanol must be metabolized into acetaldehyde in the brain. Data suggest novel targets for interventions aimed at reducing chronic alcohol intake.

Desipramine prevents stress-induced changes in depressive-like behavior and hippocampal markers of neuroprotection.

Extracellular signal-regulated kinases (ERKs) are widely implicated in multiple physiological processes. Although ERK1/2 has been proposed as a common mediator of antidepressant action in naive rodents, it remains to be determined whether the ERK1/2 pathway plays a role in depressive disorder. Here, we investigated whether chronic restraint stress (14 days) and antidepressant treatment [desipramine (DMI), 10 mg/kg intraperitoneally] induce changes in animal behavior and hippocampal levels of phospho-ERK1/2 and its substrate phospho-cAMP response element-binding protein (CREB). The results indicated that stress-induced depressive-like behaviors were correlated with an increase in P-ERK1/2 and P-CREB in the hippocampus evaluated by immunoblot analysis. As an indication of CREB activity, we evaluated changes in mRNA levels of its target genes. Brain-derived neurotrophic factor (BDNF) mRNA was reduced by stress, an effect prevented by DMI only in the CA3 area of hippocampus. Bcl-2 mRNA was reduced in all hippocampal regions by stress, an effect independent of DMI treatment. However, immunoblot from hippocampal extracts revealed that stress increased BCL-2 levels, an effect prevented by chronic DMI. These results suggest that ERKs and BDNF may be altered in depressive disorder, modifications that are sensitive to DMI action. In contrast, the stress-induced increase in BCL-2 may correspond to a neuroprotective response.

Plasticity of hippocampus following perinatal asphyxia: Effects on postnatal apoptosis and neurogenesis

Asphyxia during delivery produces long‐term deficits in brain development, including hippocampus. We investigated hippocampal plasticity after perinatal asphyxia, measuring postnatal apoptosis and neurogenesis. Asphyxia was performed by immersing rat fetuses with uterine horns removed from ready‐to‐deliver rats into a water bath for 20 min. Caesarean‐delivered pups were used as controls. The animals were euthanized 1 week or 1 month after birth. Apoptotic nuclear morphology and DNA breaks were assessed by Hoechst and TUNEL assays. Neurogenesis was estimated by bromodeoxyuridine/MAP‐2 immunocytochemitry, and the levels and expression of proteins related to apoptosis and cell proliferation were measured by Western blots and in situ hybridization, respectively. There was an increase of apoptosis in CA1, CA3, and dentate gyrus (DG) and cell proliferation and neurogenesis in CA1, DG, and hilus regions of hippocampus 1 week after asphyxia. The increase of apoptosis in CA3 and cell proliferation in the suprapyramidal band of DG was still observed 1 month following asphyxia. There was an increase of BAD, BCL‐2, ERK2, and bFGF levels in whole hippocampus and bFGF expression in CA1 and CA2 and hilus at P7 and P30. There was a concomitant decrease of phosphorylated‐BAD (Ser112) levels. The increase of BAD levels supports the idea of delayed cell death after perinatal asphyxia, whereas the increases of BCL‐2, ERK2, and bFGF levels suggest the activation of neuroprotective and repair pathways. In conclusion, perinatal asphyxia induces short‐ and long‐term regionally specific plastic changes, including delayed cell death and neurogenesis, involving pro‐ and antiapoptotic as well as mitogenic proteins, favoring hippocampal functional recovery.

Comparison of the antidepressant sertraline on differential depression-like behaviors elicited by restraint stress and repeated corticosterone administration

Depressive disorder involves emotional, cognitive, autonomic and endocrine alterations and also evidences support the role of stress in the development of this disorder. Because the hypothalamic-pituitary-adrenal axis is involved in the stress response with a concomitant rise in plasma corticoids, the present study compares the antidepressant effects of sertraline (10mg/kg, i.p.) on behavioral changes elicited by (i) restraint stress (2.5h/day for 13days) and (ii) corticosterone injections (30mg/kg, s.c., for 13days). Stressed animals, but not corticosterone-treated animals displayed anxiety behavior and a reduction in the acquisition of a conditioned avoidance response to 25% of control levels (8.0±2.2 vs. 31.7±3.2), being this effect partly sensitive to sertraline. Stressed, but not corticosterone-treated, animals displayed an increased escape failure compared with the control group (24.6%±3.5 vs. 1.6±0.7), an effect partly prevented by sertraline treatment (7.3%±2.0). Both stressed rats and corticosterone-treated rats showed an increase in immobility in the forced swim test, an effect prevented by sertraline. These results suggest that the altered behaviors elicited by stress and corticosterone can be explained by neural modifications that are sensitive to the sertraline antidepressant.

Pathophysiology of perinatal asphyxia: can we predict and improve individual outcomes?

Perinatal asphyxia occurs still with great incidence whenever delivery is prolonged, despite improvements in perinatal care. After asphyxia, infants can suffer from short- to long-term neurological sequelae, their severity depend upon the extent of the insult, the metabolic imbalance during the re-oxygenation period and the developmental state of the affected regions. Significant progresses in understanding of perinatal asphyxia pathophysiology have achieved. However, predictive diagnostics and personalised therapeutic interventions are still under initial development. Now the emphasis is on early non-invasive diagnosis approach, as well as, in identifying new therapeutic targets to improve individual outcomes. In this review we discuss (i) specific biomarkers for early prediction of perinatal asphyxia outcome; (ii) short and long term sequelae; (iii) neurocircuitries involved; (iv) molecular pathways; (v) neuroinflammation systems; (vi) endogenous brain rescue systems, including activation of sentinel proteins and neurogenesis; and (vii) therapeutic targets for preventing or mitigating the effects produced by asphyxia.

Perinatal asphyxia: current status and approaches towards neuroprotective strategies, with focus on sentinel proteins

Delivery is a stressful and risky event menacing the newborn. The mother-dependent respiration has to be replaced by autonomous pulmonary breathing immediately after delivery. If delayed, it may lead to deficient oxygen supply compromising survival and development of the central nervous system. Lack of oxygen availability gives rise to depletion of NAD+ tissue stores, decrease of ATP formation, weakening of the electron transport pump and anaerobic metabolism and acidosis, leading necessarily to death if oxygenation is not promptly re-established. Re-oxygenation triggers a cascade of compensatory biochemical events to restore function, which may be accompanied by improper homeostasis and oxidative stress. Consequences may be incomplete recovery, or excess reactions that worsen the biological outcome by disturbed metabolism and/or imbalance produced by over-expression of alternative metabolic pathways. Perinatal asphyxia has been associated with severe neurological and psychiatric sequelae with delayed clinical onset. No specific treatments have yet been established. In the clinical setting, after resuscitation of an infant with birth asphyxia, the emphasis is on supportive therapy. Several interventions have been proposed to attenuate secondary neuronal injuries elicited by asphyxia, including hypothermia. Although promising, the clinical efficacy of hypothermia has not been fully demonstrated. It is evident that new approaches are warranted. The purpose of this review is to discuss the concept of sentinel proteins as targets for neuroprotection. Several sentinel proteins have been described to protect the integrity of the genome (e.g. PARP-1; XRCC1; DNA ligase IIIα; DNA polymerase β, ERCC2, DNA-dependent protein kinases). They act by eliciting metabolic cascades leading to (i) activation of cell survival and neurotrophic pathways; (ii) early and delayed programmed cell death, and (iii) promotion of cell proliferation, differentiation, neuritogenesis and synaptogenesis. It is proposed that sentinel proteins can be used as markers for characterising long-term effects of perinatal asphyxia, and as targets for novel therapeutic development and innovative strategies for neonatal care.

Effects of perinatal asphyxia on cell survival, neuronal phenotype and neurite growth evaluated with organotypic triple cultures

Summary.The effect of perinatal asphyxia on brain development was studied with organotypic cultures from substantia nigra, neostriatum and neocortex. Asphyxia was induced by immersing foetuses-containing uterine horns removed from ready-to-deliver rats into a water bath for 20 min. Following asphyxia, the pups were nursed by a surrogate dam and sacrificed after three days for preparing organotypic cultures. Non-asphyxiated caesarean-delivered pups were used as controls. Morphological features and cell viability were recorded during in vitro development. At day in vitro (DIV) 24, the cultures were treated for immunocytochemistry using antibodies against the N-methyl-D-aspartate receptor subunit 1 (NR1) and tyrosine hydroxylase (TH).While in vitro survival was similar in cultures from both asphyxiated and control animals, differences were observed when the neuronal phenotype was assessed. Compared to controls, the total number of NR1-positive neurons in substantia nigra, as well as the number of secondary to higher level branching of TH-positive neurites from asphyxiated pups were decreased, illustrating the vulnerability of the dopaminergic systems to perinatal asphyxia.

Perinatal asphyxia: CNS development and deficits with delayed onset

Perinatal asphyxia constitutes a prototype of obstetric complications occurring when pulmonary oxygenation is delayed or interrupted. The primary insult relates to the duration of the period lacking oxygenation, leading to death if not re-established. Re-oxygenation leads to a secondary insult, related to a cascade of biochemical events required for restoring proper function. Perinatal asphyxia interferes with neonatal development, resulting in long-term deficits associated to mental and neurological diseases with delayed clinical onset, by mechanisms not yet clarified. In the experimental scenario, the effects observed long after perinatal asphyxia have been explained by overexpression of sentinel proteins, such as poly(ADP-ribose) polymerase-1 (PARP-1), competing for NAD+ during re-oxygenation, leading to the idea that sentinel protein inhibition constitutes a suitable therapeutic strategy. Asphyxia induces transcriptional activation of pro-inflammatory factors, in tandem with PARP-1 overactivation, and pharmacologically induced PARP-1 inhibition also down-regulates the expression of proinflammatory cytokines. Nicotinamide has been proposed as a suitable PARP-1 inhibitor. Its effect has been studied in an experimental model of global hypoxia in rats. In that model, the insult is induced by immersing rat fetus into a water bath for various periods of time. Following asphyxia, the pups are delivered, treated, and nursed by surrogate dams, pending further experiments. Nicotinamide rapidly distributes into the brain following systemic administration, reaching steady state concentrations sufficient to inhibit PARP-1 activity for several hours, preventing several of the long-term consequences of perinatal asphyxia, supporting the idea that nicotinamide constitutes a lead for exploring compounds with similar or better pharmacological profiles.

Corticosterone differentially regulates bax, bcl-2 and bcl-x mRNA levels in the rat hippocampus

It has previously been shown that adrenalectomy (ADX) produces apoptosis in the granule cell of the dentate gyrus (DG), and that this effect is prevented by corticosterone replacement. Thus, we have investigated how this phenomenon takes place in rat hippocampus using in situ hybridization. The expression of the pro-apoptotic gene bax was measured in the pyramidal cell fields and in the DG. After 5 days of ADX, there was a significant increase in bax mRNA levels in the suprapyramidal layer of the DG, an effect prevented by corticosterone replacement. The mRNA of the anti-apoptotic bcl-2 gene was expressed in CA3 and DG. ADX increased bcl-2 mRNA levels, but only in the suprapyramidal layer of the DG, an effect that was prevented by corticosterone administration. It is concluded that the up-regulation of bax may explain the apoptosis observed in DG after ADX, while the bcl-2 induction may correspond to a compensatory mechanism protecting the cells from death.

Plasticity of the central nervous system (CNS) following perinatal asphyxia: Does nicotinamide provide neuroprotection?

Summary.We have investigated the idea that nicotinamide, a non-selective inhibitor of the sentinel enzyme Poly(ADP-ribose) polymerase-I (PARP-1), provides neuroprotection against the long-term neurological changes induced by perinatal asphyxia. Perinatal asphyxia was induced in vivo by immersing foetuses-containing uterine horns removed from ready-to-deliver rats into a water bath for 20 min. Sibling caesarean-delivered pups were used as controls. The effect of perinatal asphyxia on neurocircuitry development was studied in vitro with organotypic cultures from substantia nigra, neostriatum and neocortex, platted on a coverslip 3 days after birth. After approximately one month in vitro (DIV 25), the cultures were treated for immunocytochemistry to characterise neuronal phenotype with markers against the N-methyl-D-aspartate receptor subunit 1 (NR1), the dopamine pacemaker enzyme tyrosine hydroxylase (TH), and nitric oxide synthase (NOS), the enzyme regulating the bioavailability of NO. Nicotinamide (0.8 mmol/kg, i.p.) or saline was administered to asphyctic and caesarean-delivered pups 24, 48 and 72 h after birth.It was found that nicotinamide treatment prevented the effect of perinatal asphyxia on several neuronal parameters, including TH- and NOS-positive neurite atrophy and NOS-positive neuronal loss; supporting the idea that nicotinamide constitutes a therapeutic alternative for the effects produced by sustained energy-failure conditions, as occurring during perinatal asphyxia.