Stimulant effects of ethanol in adolescent Swiss mice: development of sensitization and consequences in adulthood

The adolescent period is characterized by behavioral and neurobiological changes, which might predispose adolescents to the long-term negative consequences of alcohol. For example, enhanced risks of alcohol dependence are reported when drinking is initiated early. In the present studies, we used Swiss female mice to test whether chronic ethanol injections during adolescence durably affect the sensitivity to the stimulant effects of ethanol in adulthood. In a first set of experiments, several groups of young (28 day-old) mice were daily injected with various ethanol doses (1.5 – 4 g/kg) to test for ethanol sensitization during adolescence in comparison to adult mice exposed to the same schedule of ethanol injections. The results show that young mice express much higher stimulant effects after acute ethanol injections. However, they also require higher ethanol doses than adult mice to develop a sensitization to the stimulant effects of ethanol. In a second set of experiments, 28 day-old mice were sensitized to ethanol for 14 days with high ethanol doses (2.5 or 4 g/kg) and then tested for the stimulant effects of ethanol and the development of ethanol sensitization in adulthood. The results of this second set of experiments show that mice sensitized to ethanol during their adolescence remain more sensitive to the acute stimulant effects of ethanol in adulthood, especially when high ethanol doses were administered. However, the rate of the development of a sensitization to this effect was only slightly affected relative to adult mice exposed to a chronic ethanol regimen for the first time. Together, these results indicate that adolescent mice are more sensitive to the stimulant effects of ethanol but require higher ethanol doses to develop a sensitization. However, when a sensitization develops during adolescence, these mice still experience higher ethanol stimulant effects when tested in adulthood.Peer reviewe

Concomitant Caffeine Increases Binge Consumption of Ethanol in Adolescent and Adult Mice, But Produces Additive Motor Stimulation Only in Adolescent Animals

BACKGROUND: Binge co-consumption of highly caffeinated energy drinks with alcohol (ethanol [EtOH]) has become a common practice among adolescents/young adults and has been associated with an increased incidence of hazardous behaviors. Animal models are critical in advancing our understanding the neurobehavioral consequences of this form of binge drinking. Surprisingly, virtually no work has explored caffeine and EtOH co-consumption or its long-term consequences in adolescent animals. The primary objective of the current study was to extend a previously established mouse model of voluntary binge caffeine and EtOH co-consumption to explore adolescent consumption and responses compared to adults. METHODS: Adolescent and adult male C57BL/6J mice had daily limited access to caffeine (0.03% w/v), EtOH (20% v/v), a combined EtOH/caffeine solution, or water for 14 days via the binge-like drinking paradigm, drinking-in-the-dark (DID). Home cage locomotor activity was measured during DID in a subset of mice. Following DID, all mice rested for 18 days so that adolescents reached adulthood, whereupon all mice underwent 7 days of continuous access 2-bottle choice drinking for 10% (v/v) EtOH or water. RESULTS: Co-consumption with caffeine significantly increased EtOH intake and resultant blood ethanol concentrations in both adolescent and adult mice. In addition, adolescent mice exhibited a uniquely robust locomotor stimulant response to caffeine and EtOH co-consumption. Later EtOH intake and preference was not influenced, however, by prior fluid consumption history via DID. CONCLUSIONS: Together with findings from the human literature, our results suggest that caffeine co-consumption may positively influence binge alcohol consumption in adolescents/young adults. Importantly, this age group may be particularly sensitive to the additive stimulant effects of caffeinated alcohol consumption, an effect which may be related to the high incidence of associated negative outcomes in this population. These observations are particularly concerning considering the heightened plasticity of the adolescent brain

Relationship between transcranial magnetic stimulation markers of motor control and clinical recovery in obsessive compulsive disorder/Gilles de la Tourette syndrome: a proof of concept case study

BackgroundObsessive compulsive disorder (OCD) and Gilles de la Tourette syndrome (GTS) are neurodevelopmental disorders characterized by difficulties in controlling intrusive thoughts (obsessions) and undesired actions (tics), respectively. Both conditions have been associated with abnormal inhibition but a tangible deficit of inhibitory control abilities is controversial in GTS.MethodsHere, we examined a 25 years-old male patient with severe OCD symptoms and a mild form of GTS, where impairments in motor control were central. Transcranial magnetic stimulation (TMS) was applied over the primary motor cortex (M1) to elicit motor-evoked potentials (MEPs) during four experimental sessions, allowing us to assess the excitability of motor intracortical circuitry at rest as well as the degree of MEP suppression during action preparation, a phenomenon thought to regulate movement initiation.ResultsWhen tested for the first time, the patient presented a decent level of MEP suppression during action preparation, but he exhibited a lack of intracortical inhibition at rest, as evidenced by reduced short-interval intracortical inhibition (SICI) and long-interval intracortical inhibition (LICI). Interestingly, the patient’s symptomatology drastically improved over the course of the sessions (reduced obsessions and tics), coinciding with feedback given on his good motor control abilities. These changes were reflected in the TMS measurements, with a significant strengthening of intracortical inhibition (SICI and LICI more pronounced than previously) and a more selective tuning of MEPs during action preparation; MEPs became even more suppressed, or selectively facilitated depending on the behavioral condition in which they we probed.ConclusionThis study highlights the importance of better understanding motor inhibitory mechanisms in neurodevelopmental disorders and suggests a biofeedback approach as a potential novel treatment

ESR spectroscopy for the study of an inflammation-induced AKI cellular model

The kidney is faced to an impairment of oxygen extraction during sepsis which is well-known to be a risk factor for the development of acute kidney injury (AKI). Recent research activities in the mechanisms involved in the development of AKI in sepsis emphasize the central role of hemodynamic and inflammatory events. More particularly, two mechanisms are suggested to explain the inability of the injured kidney to extract oxygen: tissue hypoxia and cellular energetic metabolism dysfunction. Our working hypothesis of the pathophysiology of AKI is based on cellular respiratory dysfunction due to the inflammatory response inherent to sepsis. To study the mechanism of oxygen regulation in inflammation-induced acute kidney injury, we investigate the effects of a bacterial endotoxin (lipopolysaccharide, LPS) on the basal respiration of proximal tubular epithelial cells (HK-2) by ESR oximetry. This method has shown that HK-2 cells exhibit a decreased oxygen consumption rate when treated with LPS. Surprisingly, this cellular respiration alteration persists even after the stress factor was removed. We suggested that this irreversible decrease in renal oxygen consumption after LPS challenge is related to a pathologic metabolic down-regulation such as a lack of oxygen utilization by cells. This decrease was accompanied by increased nitric oxide (NO) production as measured by a spin trapping technique using ESR spectroscopy. This method is based on the trapping of NO by a metal-chelator complex consisting of N-methyl-D-glucamine dithiocarbamate (MGD) and reduced iron (Fe2+) forming a water-soluble NO-FeMGD complex detected by ESR. Since inducible NO synthase (iNOS) has been shown to play an important role in sepsis-induced AKI, the iNOS inhibitor L-NMMA (L-NG-monomethyl Arginine citrate) was tested in this in vitro model. L-NMMA blocked NO generation and permitted the HK-2 cells to recover a normal cellular respiration. Overall, ESR spectroscopy and the model of HK-2 cells exposed to LPS displays some key features of inflammation-induced acute kidney injury

Endotoxin-induced alterations in renal oxygen consumption: an ESR oximetry study

The kidney, one of the most injured organs in critically ill patients, is faced with unique challenges for molecular oxygen regulation. Recent research activities in the pathophysiological mechanism of acute renal injury (ARI) emphasize the central role of hemodynamic and inflammatory events in septic shock. More particularly, two mechanisms have been postulated to explain the inability of the injured kidney to extract oxygen: tissue hypoxia and cellular energetic metabolism dysfunction. The present investigation was carried out to characterize the effects of bacterial endotoxin on the oxygen consumption of human tubular proximal cell line (PTC) by using the very sensitive electron spin resonance oximetry method. Oxygen consumption was shown to decrease quite markedly in cells treated with lipopolysaccharide (LPS) from 16.52 ± 2.51 (n=6) in the control group to: 12.94 ± 2.62 (n=3) in the short incubation time group (6h) and 10.86 ± 2.20 (n=3) in the long incubation time group (18h). This decrease in oxygen consumption in renal cells after LPS challenge may be in relation with a metabolic down-regulation. Renal energetic are deranged in sepsis not just because O2 delivery is impaired but perhaps also because the ability of cells to utilize available O2 is compromised

ESR evidence of superoxide anion, hydroxyl radical and singlet oxygen generation during photosensitization of PPME in HCT-116 cells

Pyropheophorbide-a methyl ester (PPME), a derivative of chlorophyll a, is a second-generation photosensitizer and is studying largely in vitro for nearly a decade on cancerous cells. It has been previously established on HCT-116 (human colon carcinoma cell line) that PPME is a molecule able to create apoptotic and necrotic death (Matroule et al). The cytotoxicity of PPME is presumed to be induced by reactive oxygen species (ROS) generated by the photoexcited molecule. Actually, to the best of our knowledge, no experimental evidence enables to confirm this supposition in an indubitable manner. Electron spin resonance (ESR) associated with spin trapping technique is a powerful method to detect, quantify and identify the ROS produced after photoactivation of PPME. Previous results indicate that PPME penetrates inside cells and localizes inside specific organelles (endothelial reticulum, Golgi apparatus and lysosome) (Matroule et al). The recent researches of Guelluy et al have also clearly demonstrated the presence of PPME inside mitochondrion. Consequently, ESR experiments were performed using an intracellular located spin trap, POBN (4-pyridyl 1-oxide-N-tert-butylnitrone), in order to detect in situ the ROS production. It has been shown that PPME is able to generate superoxide anions and hydroxyl radicals. Irradiation of the dye in HCT-116 cells in the presence of POBN spin trap and ethanol scavenger (2%, a non-toxic concentration) leads to the apparition of the ESR spectrum characteristics of POBN/ethoxy adduct. To assess the extent of contribution of ROS and to determine a possible reaction mechanism, competition experiments with specific quencher agents were carried out. Addition of catalase (CAT), a hydrogen peroxide quencher, or superoxide dismutase (SOD), a superoxide anion quencher, inhibits 30% of the signal. The parallel effect of SOD and CAT suggest that superoxide anion and hydrogen peroxide are involved in the generation of hydroxyl radicals via a Fenton reaction. This assertion is reinforced by the 20% reduction of signal intensity when adding desferroxamine, a Fe3+ chelator also implicated in Fenton reaction. Addition of DABCO, a quencher of singlet oxygen, to cells reduces 70% of the POBN/ethoxy adduct signal intensity.Diversification des méthodes de microscopie pour l'étude en milieu cellulaire du stress oxydatif induit - impact de la vectorisation médicamenteus

In vitro ESR measurements: powerful tool to study toxic effects on cells

Electron spin resonance spectroscopy (ESR) is a highly efficient technique able to access a wide range of information about the unfavourable effects caused by a chemical or a drug. ESR in spin labelling fits well in with the study of membranes and particularly with the changes in lipid bilayer organisation induced by drug. Our team previously developed a way to quantify the effective lipid bilayer microviscosity of cell membranes and consequently put in evidence the fluidity effect of the propofol. Recently, the importance of lipid raft domains has been shown due to their important role as a platform for signal transduction and protein sorting. We propose to highlight the effect of the Randomly methylated beta cyclodextrin (Rameb) on these domains on membrane model (liposomes) as well as on colon carcinoma cell line (HCT-116). Futhermore, ESR in spin trapping is used in order to identify and quantify the generation of Reactive Oxygen Species (ROS) in cells. An ESR study on human colon carcinoma cell line has highlighted the cytotoxicity of the photosensitizer pyrophephorbide-a methyl ester. Using an intracellular located spin trap (4-pyridyl 1-oxide-N-tert-butylnitrone, POBN), it has been shown that the photoexcitation of the dye is able to generate superoxide anions, hydroxyl radicals and singlet oxygen. Moreover, ESR is one of the most sensitive method for measuring cellular oxygen consumption. Our team has studied the alterations of oxygen respiratory in human tubular renal cells treated with an endotoxin (lipopolysaccharide, LPS). The incubation of HK-2 cells with LPS elicited a decreased in oxygen consumption suggesting a down-regulation of the cells metabolism

A confocal microscopic study of mitochondrial alterations of renal HK-2 cells exposed to an endotoxic stress

Sepsis has a profound deleterious effect on kidney functions through complex mechanisms, which involve the immune response, inflammatory pathways, intracellular dysfunction and hemodynamic instability. Those factors are difficult to discriminate in vivo. To get a better understanding of renal respiratory dysfunction, we developed an in vitro model of sepsis-induced acute kidney injury using proximal tubular epithelial cell lines (HK-2) exposed to a bacterial endotoxin (lipopolysaccharide, LPS). Using this model, our first work has demonstrated that the basal respiration of renal HK-2 cells subjected to endotoxins was altered and presented a strong decrease in the oxygen consumption rates. Our working hypothesis of the pathophysiology of sepsis-induced AKI is based on a change in mitochondrial function that has been termed cytopathic hypoxia. A consequence of mitochondrial function alterations is an inability of the cell to use molecular oxygen for ATP production. The oxidative phosphorylation within mitochondria is interrupted because of the inhibition of cytochrome oxidase. The present investigation was carried out to establish whether mitochondrial alterations might be a mechanism of renal tubular epithelial injury during sepsis. To reach this goal the mitochondrial alterations of renal HK-2 cells exposed to an endotoxic stress was studied by confocal laser-scanning microscope. Confocal microscope allowed observation of the evoked phenomena at the single cell level and in real time. More particulary, mitochondrial morphology, mitochondrial membrane potential (ΔΨm) and generation of reactive oxygen species were recorded using specific vital fluorescent probes and quantified by image processing and analysis. Mitochondrial membrane potential is generated by the mitochondrial electron transport chain. This gradient is critical for the formation of ATP, and a fall in membrane potential is an indicator of mitochondrial dysfunction. ΔΨm was measured using the lipophilic cationic probe TMRE and it was shown that LPS produced a decrease in ΔΨm. In parallel, superoxide generation was measured by using MitoSOX which is selectively targeted to the mitochondria. There was a significant increase in mitochondrial superoxide-specific oxidation of MitoSOX when HK-2 cells were submitted to LPS. Overall, the model of HK-2 cells exposed to LPS displays some key features of sepsis-induced acute kidney injury. The confocal microscopy study has suggested a mechanism of toxicity dependent on mitochondrial oxidant generation and mitochondrial dysfunction. Indeed, the exposure to LPS has resulted in an increased generation of superoxide and a loss of mitochondrial function probably initiated by a fall in mitochondrial potential

Etude de l'ontogenèse des effets comportementaux et toxicomanogènes de l'alcool chez la souris

Adolescence is a critical period of development, characterized by a particular vulnerability to the consequences of drug abuse, including alcohol. Although the mechanisms underlying this enhanced vulnerability are not clearly identified, the increased cerebral plasticity of this stage of develompent seems to be a major contributor. The main aim of the present project was to precisely characterize the development of the sensitivity to the different behavioral effects of ethanol as well as the evolution in alcohol vulnerability across the different phases of adolescence and adulthood. These effects were tested in mice, in which different ontogenetic phases were clearly established. Our results have shown that the stimulant effects of ethanol gradually decreased during adolescence, whereas its sedative effects increased with age. Moreover, higher doses were required to induce behavioral sensitization to the stimulant effects of ethanol in adolescent mice. However, with effective ethanol doses, younger mice developed very significant sensitization. Finally, chronic alcohol exposure during adolescence durably enhanced the sensitivity to the stimulant effects of ethanol and their sensitization, while it reduced the sensitivity to its sedative effects. In conclusion, this work reveals that adolescents might be especially sensitive to the positive effects of alcohol. Furthermore, chronic alcohol consumption during adolescence could increase the risk of later alcohol-related disorders./L'adolescence est une période de développement critique, également caractérisée par une vulnérabilité particulière aux risques d'abus d'alcool. Cette vulnérabilité accrue est en partie expliquée par la plus grande plasticité cérébrale des adolescents, bien que les mécanismes précis ne soient pas encore clairement élucidés. L’objectif de ce travail est de caractériser finement l'évolution ontogénétique des effets comportementaux de l’éthanol et de la vulnérabilité à l'abus d'alcool lors des différents sous-stades de l'adolescence et à l'âge adulte en utilisant comme modèle animal la souris, pour laquelle différents stades ontogénétiques ont été clairement établis. Nos résultats démontrent que la sensibilité aux effets stimulants de l’éthanol diminue graduellement au cours de l’adolescence, tandis que la sensibilité à ses effets sédatifs augmente. De plus, l’induction d’une sensibilisation comportementale aux effets stimulants de l’éthanol à l’adolescence requiert l’administration répétée de doses plus élevées. Néanmoins, lors d’une exposition chronique aux doses adéquates, les adolescents développent une sensibilisation plus forte qu’à l’âge adulte. Enfin, une exposition répétée à des doses élevées d’éthanol au cours de l’adolescence accroît durablement la sensibilité aux effets stimulants aigus de l’alcool et à leur sensibilisation, tandis que la sensibilité à son action sédative diminue. En conclusion, ce travail indique que les adolescents semblent particulièrement sensibles aux effets positifs de l’éthanol. De plus, une consommation répétée d’alcool à l’adolescence accroitrait durablement le risque de connaître des troubles d’abus et de dépendance alcoolique