Diego Bustamante

Antinociceptive effects of Ca2+ channel blockers

The antinociceptive action of four Ca2+ channel blockers, nifedipine, nimodipine, verapamil and diltiazem, was evaluated and compared to that of morphine using three algesiometric tests in mice and rats, namely, formalin, writhing and modified hot-plate test. Dose-response curves for all the drugs tested were similar and a significant dose-dependent antinociceptive action was evident in the formalin and writhing tests. However, in the hot-plate test, only nimodipine exhibited a significant analgesic effect, confirming the misleading results previously reported for this test. The findings suggest a pharmacological role of Ca2+ channel blockers in the modulation of antinociception under acute conditions. The analgesic action of Ca2+ channel blockers could be mediated by an increase in the nociceptive threshold resulting from interference with Ca2+ influx at opioid receptors, because Ca2+ influx is critical for the release of neurotransmitters and other substances implicated in nociception and inflammation. It is suggested that if a substance has a Ca2+ channel blocking effect, it should probably have some antinociceptive properties.

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.

Effect of single and repeated methamphetamine treatment on neurotransmitter release in substantia nigra and neostriatum of the rat.

The main purpose of this study was to characterize the initial neurotransmission cascade elicited by methamphetamine, analysing simultaneously with in vivo microdialysis monoamine, amino acid and neuropeptide release in substantia nigra and neostriatum of the rat. The main effect of a single systemic dose of methamphetamine (15 mg/kg, subcutaneously) was an increase in dopamine levels, both in substantia nigra (≈ 10‐fold) and neostriatum (≈ 40‐fold), accompanied by a significant, but lesser, increase in dynorphin B (≈ two‐fold, in both regions), and a decrease in monoamine metabolites. A similar effect was also observed after local administration of methamphetamine (100 µm) via the microdialysis probes, but restricted to the treated region. In other experiments, rats were repeatedly treated with methamphetamine or saline, with the last dose administered 12 h before microdialysis. Dopamine K+‐stimulated release was decreased following repeated methamphetamine administration compared with that following saline, both in the substantia nigra (by ≈ 65%) and neostriatum (by ≈ 20%). In contrast, the effect of K+‐depolarization on glutamate, aspartate and GABA levels was increased following repeated administration of methamphetamine. In conclusion, apart from an impairment of monoamine neurotransmission, repeated methamphetamine produces changes in amino acid homeostasis, probably leading to NMDA‐receptor overstimulation.

Stress promotes development of ovarian cysts in rats : The possible role of sympathetic nerve activation ()

AbstractActivation of the sympathetic innervation precedes the induction of polycystic ovaries in rats given estradiol valerate (EV). The mechanism of induction by EV may thus involve both direct and neurogenic components. We tested this hypothesis using a combined cold and restraint stress to induce an increase in sympathetic tone, including that of the ovarian sympathetic nerves. Three weeks after the start of stress we found:1.An increase in the content of norepinephrine (NE) in the celiac ganglion.2.An increase in the release of NE from the ovary.3.An unchanged NE uptake by the ovary.4.An unchanged content of NE in the ovary. The ovarian content of neuropeptide Y (NPY) (colocalized with NE) was significantly decreased. These results suggest that NE synthesis and its secretion are increased during this period and correlate with the increase in secretion of androgens and estradiol, the development of precystic follicles, and a decrease in the ovulatory rate. After 11 wk, NE release had returned to control values, whereas the ovarian NE content had risen significantly, suggesting a maintained high rate of NE synthesis. In the ovary, NPY contents, steroid secretion, morphology, and ovulation had returned to the control state. These results suggest the participation of an extraovarian factor that might act locally to control the release of NE from the ovary, and further support the hypothesis that increased sympathetic activity plays a role in the development and maintenance of ovarian cysts.

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.

Ethanol induces stronger dopamine release in nucleus accumbens (shell) of alcohol-preferring (bibulous) than in alcohol-avoiding (abstainer) rats

Several studies on the differences between ethanol-preferring versus non-preferring rat lines suggest an innate deficit in the mesolimbic dopaminergic system as an underlying factor for ethanol volition. Rats would try to overcome such deficit by engaging in a drug-seeking behaviour, when available, to drink an ethanol solution over water. Thus, in the present study we compared the effect of a single dose of ethanol (1 g/kg, i.p.) on the extracellular levels of monoamines measured by microdialysis in the shell of nucleus accumbens of University of Chile bibulous (UChB) and University of Chile Abstainer (UChA) rats, bred for 79 and 88 generations to prefer or reject ethanol, respectively. It is reported that under basal conditions extracellular dopamine levels are lower in the bibulous than in the abstainer rats, while ethanol induced a 2-fold greater increase of dopamine release in bibulous than in abstainer rats. The greater effect of ethanol in bibulous rats was not associated to differences in blood ethanol levels, since the concentration and elimination of ethanol were virtually identical in both rat lines, indicating that bibulous rats are more sensitive to the stimulation of dopamine release by ethanol than abstainer rats. No differences were observed in 5-hydroxytryptamine or metabolites measured simultaneously under basal or ethanol-stimulating conditions in bibulous and abstainer rats. Overall, the present results suggest that a low dopaminergic tone and a strong mesolimbic dopamine response to ethanol are concerted neurochemical features associated to an ethanol-seeking behaviour in rats.

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.

Progesterone and Testosterone Modulate the Convulsant Actions of Pentylenetetrazol and Strychnine in Mice

The influence of progesterone and testosterone on the incidence of seizures after administration of intraperitoneal pentylenetetrazol and subcutaneous strychnine was evaluated in mice. Pentylenetetrazol and strychnine were administered in doses that induced seizures in 40-50% of control mice in dioestrus (48 and 0.9 mg/kg, respectively). The percentage of seizures induced by pentylenetetrazol and strychnine was significantly lower in female mice in prooestrus/oestrus, when progesterone levels are high, than in dioestrus, when progesterone levels are low. Pretreatment of pentylenetetrazol-challenged mice with progesterone (250 microg/kg) increased the incidence of seizures in prooestrus/oestrus, without affecting seizures in dioestrus. The same pretreatment in strychnine-challenged mice also increased the incidence of seizures in prooestrus-dioestrus, but significantly reduced the incidence of seizures in dioestrus. In addition, progesterone pretreatment significantly increased the percentage of deaths induced by strychnine in prooestrus-oestrus, reducing deaths in dioestrus. Orchidectomized male mice had a significantly higher incidence of seizures after administration of pentylenetetrazol and strychnine than control mice. Administration of 11 daily doses of 250 microg/kg of testosterone to castrated mice significantly reduced the incidence of seizures induced by pentylenetetrazol. These results confirm the modulatory influence of reproductive steroids on the excitability of the central nervous system and the possible clinical importance of progesterone and testosterone in the management of partial epilepsy.

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.