Maria del Carmen Sanchez-Amate

Individual differences in schedule-induced polydipsia and the role of gabaergic and dopaminergic systems

RationaleThe research of individual differences has opened new possibilities for better exploring the neurobiological basis of vulnerability to psychopathological disorders.ObjectiveWe extended this approach by using schedule-induced polydipsia (SIP).MethodsOutbred male Wistar rats were characterized as either high (HD) or low (LD) drinker according to their behavior in SIP. Subsequently, their performance in the elevated plus maze (EPM) was studied for possible differences in anxiety-like behaviors. Finally, the effects of pentylenetetrazol (PTZ), diazepam, d-amphetamine, and cocaine on individual differences in SIP were investigated.ResultsHD rats acquired SIP faster and reached higher asymptotic levels than LD. Nose pokes, however, were greater in LD. In the EPM, there were no differences between HD and LD animals. Gabaergic drug effects on SIP did not differ between HD and LD rats. Compared to saline, PTZ reduced and diazepam increased water SIP drinking. On the other hand, amphetamine dose-dependently reduced SIP in HD, whereas the highest dose was required to reduce SIP in LD. HD rats also showed reductions in SIP drinking after cocaine administration. However, the effects of these drugs on nose pokes did not differ between HD and LD.ConclusionThese data provide novel evidence that individual differences in SIP are not predictive of behavioral reactivity in animal models of anxiety and suggest an important role for the dopaminergic system in such individual differences. These findings point to SIP as a useful animal model for investigating the neurobiological basis of vulnerability to several psychopathologies in which the dopaminergic system is involved.

Studies on Phospholipid Biosynthesis in Hepatocytes from Alcoholic Rats by Using Radiolabeled Exogenous Precursors

We have studied the synthesis of phospholipids in hepatocytes isolated from chronically ethanol-treated rats by using isotopically labelled serine, ethanolamine, and choline as exogenous precursors. Our results demonstrate that ethanol induces specific effects on the biosynthesis of phosphatidyl-ethanolamine and phosphatidylcholinevia CDP-derivatives and also on the synthesis of phosphatidylserinevia the Ca++-dependent base-exchange reaction. Thus, the synthesis of phosphatidylethanolamine from [3-H]ethanolamine and the incorporation of [3H]serine into phosphatidylserine were clearly higher in hepatocytes from ethanol-treated rats compared to controls. The synthesis of phosphatidylcholine from [methyl-14C] choline, on the other hand, decreased markedly, suggesting a specific inhibition of cholinephosphotransferase activity. We have also demonstrated that the phosphatidylcholine levels are markedly decreased in hepatocytes isolated from chronically ethanol-treated rats as a consequence of the lower phosphatidylcholine biosynthesis. The decrease in the incorporation of radioactivity from choline to betaine, which we also found, is interpreted as being the result of a higher use of betaine as methyl donor instead of methionine to maintain the hepaticS-adenosylmethionine levels in chronic alcoholism.

Evidence of differential effects produced by ethanol on specific phospholipid biosynthetic pathways in rat hepatocytes

1 The aim of the present study was to investigate the effects of ethanol in vitro on the phospholipid biosynthetic pathways in hepatocytes isolated from the rat. We have used [methyl‐14C]‐choline, [1‐3H]‐ethanolamine and L‐[3‐3H]‐serine as exogenous precursors of the corresponding phospholipids, phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS). 2 Incubation of hepatocytes in the presence of ethanol significantly alters the incorporation of radiolabel from [14C]‐choline and [3H]‐ethanolamine into the metabolic intermediates and the final products of the CDP‐choline and CDP‐ethanolamine pathways. Radioactivity in the metabolic intermediates of both pathways was significantly decreased and the amount of label in PE was reduced whilst that of PC was not modified. 3 In the presence of 4‐methylpyrazole, an inhibitor of alcohol dehydrogenase (ADH) activity, ethanol produces a reduction in the label of choline phosphate, ethanolamine phosphate and a significant decrease in the amount of PC and PE radiolabel. 4 On the other hand, ethanol increases the incorporation of serine into phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine, although this effect is observed only in the absence of 4‐methylpyrazole, indicating that this alteration is produced by some metabolite generated as a consequence of hepatic alcohol metabolism. 5 Ethanol also interferes with the methylation of phosphatidylethanolamine produced via the CDP‐ethanolamine pathway but it does not alter phosphatidylethanolamine methylation when this phospholipid is produced by mitochondrial phosphatidylserine decarboxylation, suggesting the existence of different intramembrane pools of phosphatidylethanolamine, which may exhibit different sensitivity to alcohol. 6 Our results indicate that ethanol exerts two different effects on phospholipid metabolism in hepatocytes: a stimulatory effect on the incorporation of exogenous substrates into different phospholipids probably related to an alteration in the availability of lipogenic substrates as a consequence of ethanol metabolism, and another inhibitory effect produced by ethanol per se, which can be observed only when ethanol metabolism is inhibited by the presence of a specific inhibitor of alcohol dehydrogenase activity.

Orexin receptor 1 signaling contributes to ethanol binge-like drinking: Pharmacological and molecular evidence.

Orexins (OX) have been recently implicated in ethanol seeking and self-administration. A few recent studies have provided additional evidence that OX receptor antagonists effectively reduce voluntary ethanol consumption in subjects spontaneously showing high levels of ethanol intake. The present study further evaluates the contribution of OXR1 to excessive binge-like drinking of ethanol in ad libitum-fed C57BL/6J mice from a pharmacological and molecular approach. The main findings in the study are: (1) Icv administration of SB-334867 (3 μg/μl) blunted ethanol (20% v/v), but not saccharin (0.15% w/v) binge-like drinking in a drinking in the dark procedure, without any alteration of chow consumption or total calories ingested; (2) Icv administration of SB-334867 (3 μg/μl) increased the latency to recover the righting reflex after a sedative dose of ethanol without any significant alteration in ethanol peripheral metabolism; (3) four repetitive, 2-h daily episodes of saccharin, but not ethanol binge-like drinking blunted OXR1 mRNA expression in the lateral hypothalamus. Present findings extend the current knowledge pointing to a role for OX signaling in ethanol sedation, which might partially explain the inhibitory effect of OXR1 antagonists on ethanol consumption. Combined pharmacological and molecular data suggesting the contribution of OXR1 in ethanol binge-drinking leading us to propose the idea that targeting OXR1 could represent a novel pharmacological approach to control binge-consumption episodes of ethanol in vulnerable organisms failing to spontaneously reduce OX activity.

Acute high dose of chlorpyrifos alters performance of rats in the elevated plus-maze and the elevated T-maze

Chlorpyrifos (CPF) is a broad spectrum organophosphate (OP) pesticide widely used in agriculture, industry and household. Several animal studies indicate emotional disturbances after CPF exposure, although the results are sometimes puzzling. Thus, both anxiolytic and anxiogenic effects of CPF have been reported in different animal models of anxiety [Sánchez-Amate MC, Flores P, Sánchez-Santed F. Effects of chlorpyrifos in the plus-maze model of anxiety. Behav Pharmacol 2001;12:285-92; Sánchez-Amate MC, Dávila E, Cañadas F, Flores P, Sánchez-Santed F. Chlorpyrifos shares stimulus properties with pentilenetetrazol as evaluated by and operant drug discrimination task. Neurotoxicology 2002;23:795-803; López-Crespo G, Carvajal F, Flores P, Sánchez-Santed F, Sánchez-Amate MC. Time-course of biochemical and behavioural effects of a single high dose of chlorpyrifos. Neurotoxicology 2007;28:541-7]. On the other hand, other behavioural effects of CPF are time-dependent [López-Crespo G, Carvajal F, Flores P, Sánchez-Santed F, Sánchez-Amate MC. Time-course of biochemical and behavioural effects of a single high dose of chlorpyrifos. Neurotoxicology 2007;28:541-7], raising the question that the effects of CPF could be task and post-administration time dependent. To test this hypothesis, three groups of rats were treated with a single high dose of CPF (250 mg/kg); one of the groups was tested on day 5 on the elevated plus-maze, to complete our previous study on day 2 [Sánchez-Amate MC, Flores P, Sánchez-Santed F. Effects of chlorpyrifos in the plus-maze model of anxiety. Behav Pharmacol 2001;12:285-92]. The remaining groups were tested on the elevated T-maze on days 2 and 5. CPF produced an increased open arm activity on the elevated plus-maze on day 5, an increased escape latency on the elevated T-maze on day 2 and an impaired inhibitory avoidance on day 5. Data are discussed taking together all studies carried out in our laboratory, confirming that CPF effects on emotional behaviour are dependent on both task contingencies and post-administration time.

Adaptive changes induced by chronic ethanol ingestion on hepatic mitochondrial and microsomal enzyme activities

Abstract Ethanol exerts its pharmacological effects by altering the physico-chemical properties of biological membranes. Modifications induced by ethanol may result in changes in the activity of membrane-bound enzymes that have been shown to require a specific membrane fluidity and composition for optimal function. In this study we have carried out the analysis of the effects of ethanol on the different enzyme activities of hepatic microsomes and mitochondria. Our results show that chronic ethanol treatment causes marked changes to enzyme activity in the mitochondrial and microsomal electron-transport systems in chick liver. The inhibition observed in the mitochondrial enzyme activities studied, indicates that ethanol ingestion depresses the functionality of the respiratory-chain. In microsomes, NADH cytochrome c reductase activity was significantly decreased whilst NADH cytochrome b5 reductase activity was increased after ethanol administration. Hepatic mitochondria and microsomes from control and chronic ethanol-treated chicks were submitted to ethanol in vitro in order to study the possible existence of adaptive changes in the different enzyme systems as consequence of long-term ethanol administration. Incubation of control membranes with different amounts of ethanol induced marked alterations in enzyme activities. In membranes isolated from ethanol-treated chicks, ethanol also produced a similar effect except with cytochrome oxidase and NADH cytochrome b5 reductase, which resisted alteration by ethanol added, suggesting the existence of adaptive changes in these enzyme activities that allow them to remain unaltered after exposure to ethanol in vitro.

Repeated Binge-Like Ethanol Administration During Adolescence cause Decreased C-Fos Immunoreactivity in Amygdala and Arcuate Nucleus in Adult Sprague-Dawley Rats

El consumo en atracon durante la adolescencia esta asociado con neurotoxicidad y con el riesgo de desarrollar un trastorno en el uso de alcohol. Diversos estudios muestran que la administracion aguda y cronica de alcohol en ratas adultas altera la expresion de c-fos, un marcador indirecto de actividad celular, en diferentes areas cerebrales. Nosotros evaluamos si el patron de consumo de alcohol en atracon durante la adolescencia tiene un impacto en la actividad basal de c-fos en esas regiones activadas por el alcohol. Utilizamos ratas Sprague-Dawley en su dia post-natal 25 (PND25) tratadas con suero salino (grupo SP) o con etanol tipo atracon (grupo BEP) durante dos dias consecutivos, en intervalos de 48 h, durante 14 dias (PND25- PND38). En la edad adulta (PND63) y despues de 25 dias sin etanol, evaluamos la inmunorreactividad para c-fos en respuesta a una administracion aguda de suero salino o etanol (1,5 o 3,0 g/kg) en diferentes regiones cerebrales. La administracion de alcohol incremento de manera dosis-dependiente la actividad de c-fos en el nucleo paraventricular del hipotalamo. Ademas la exposicion a etanol tipo atracon durante la adolescencia disminuyo la actividad basal de c-fos en la adultez en el nucleo central de la amigdala y en el nucleo arqueado del hipotalamo. Concluimos que el consumo de alcohol en atracon durante la adolescencia causa problemas a largo plazo en la actividad basal de regiones cerebrales implicadas en el consumo de alcohol.

Interaction Between Exposure to Neurotoxicants and Drug Abuse

Humans are continuously exposed to a variety of environmental neurotoxicants. Over the past 30 years, at least 100,000 chemicals, including pesticides, food additives, drugs, and cosmetics, have been registered for commercial use in the United States (Muir & Howard, 2006). Twenty years ago, about 750 chemicals had shown neurotoxic effects in laboratory animals (Anger, 1984). Actually, the number is thought to exceed a thousand, although no authoritative estimate of the real number of neurotoxicants is available (Grandjeand & Landrigan, 2006).

A Single High Dose of Chlorpyrifos Reduces Long-Term Basal C-Fos Expression in the Rat Arcuate Hypothalamic Nucleus

El clorpirifos (CPF) es un compuesto organofosforado utilizado como plaguicida en todo el mundo. Despues de ser inyectado de manera subcutanea, mantiene la actividad de la enzima acetilcolinesterasa (AChE) inhibida durante meses. Estudios clinicos y experimentales muestran que la exposicion al CPF induce deficits neuroconductuales persistentes en seres humanos y animales, incluso despues de un solo episodio/inyeccion. Ademas, estudios epidemiologicos evidencian que un porcentaje significativo (60%) de los veteranos de la Guerra del Golfo, asi como los agricultores que sufren una intoxicacion aguda por organofosforados, desarrollan intolerancia a la nicotina y las bebidas que contienen etanol. Datos experimentales mostraron que la administracion de una sola dosis alta de CPF en ratas Wistar adultas provoca una reduccion a largo plazo del consumo voluntario de etanol y un incremento en la sedacion provocada por etanol sin evidencias de alteracion del metabolismo de esta sustancia, lo que indica que pueden existir interacciones neurobiologicas entre CPF-etanol. En este estudio, se explora si la exposicion a CPF induce alteraciones significativas en la actividad neuronal basal o evocada por el etanol en un conjunto de regiones colinoceptivas del cerebro involucradas en las respuestas neurobiologicas al etanol. Para ello, se evaluo la expresion de c-fos en respuesta a una dosis de etanol aguda (1.5 o 3.0 g/kg, ip) o solucion salina en ratas Wistar macho adultas previamente inyectados con dosis aguda de CPF (250 mg/kg, sc) o un vehiculo. Encontramos que la administracion de CPF redujo la expresion basal de c-fos a largo plazo, pero no la evocada por el etanol en el nucleo arqueado del hipotalamo. Debido a que vias cerebrales independientes podrian modular las respuestas agudas al etanol y el consumo voluntario del mismo, no se descarta la contribucion de las alteraciones neuronales basales observadas en el Arc en la evitacion del consumo de etanol provocado por CPF.