Long-lasting teratogenic effects of nicotine on cognition: gender specificity and role of AMPA receptor function.

Nicotine, the main psychoactive ingredient in tobacco, readily crosses the placental barrier to cause growth and neurobehavioral abnormalities in the offspring. The current study was designed to assess whether nicotinic action causes long lasting teratogenic effects and synaptic dysfunctions. Pregnant Sprague-Dawley rats were infused with nicotine via osmotic minipumps at a dose of 6 mg/kg/day corresponding to the dose receiving during heavy smoking. A battery of behavioral tests and electrophysiological experiments were performed during specific postnatal periods. A spectrum of developmental and behavioral modifications in adolescent, young-adult and aged animals resulted after prenatal nicotine exposure. The potentially teratogenic effect of nicotine was clearly demonstrated in both genders by changes in developmental reflexes, exploratory and novelty seeking behavior, as well as a higher level of anxiety, and changes in individual and group responses in learning and memory. Most of the behavioral abnormalities were transitional with advancing age (6 months), although cognitive deficits measured by a two-way active avoidance task were long-lasting for male rats. Electrophysiological studies show decreased excitatory postsynaptic responses (mEPSCs) mediated by AMPA receptors in the hippocampus. These results suggest that teratogenic effect of nicotine on cognition is age and gender-specific, long-lasting and associated with AMPA receptor function.

Distribution of alcohol dehydrogenase mRNA in the rat central nervous system. Consequences for brain ethanol and retinoid metabolism.

The localization of alcohol dehydrogenase (ADH) in brain regions would demonstrate active ethanol metabolism in brain during alcohol consumption, which would be a new basis to explain the effects of ethanol in the central nervous system. Tissue sections from several regions of adult rat brain were examined by in situ hybridization to detect the expression of genes encoding ADH1 and ADH4, enzymes highly active with ethanol and retinol. ADH1 mRNA was found in the granular and Purkinje cell layers of cerebellum, in the pyramidal and granule cells of the hippocampal formation and in some cell types of cerebral cortex. ADH4 expression was detected in the Purkinje cells, in the pyramidal and granule cells of the hippocampal formation and in the pyramidal cells of cerebral cortex. High levels of ADH1 and ADH4 mRNAs were detected in the CNS epithelial and vascular tissues: leptomeninges, choroid plexus, ependymocytes of ventricle walls, and endothelium of brain vessels. Histochemical methods detected ADH activity in rodent cerebellar slices, while Western-blot analysis showed ADH4 protein in homogenates from several brain regions. In consequence, small but significant levels of ethanol metabolism can take place in distinct areas of the CNS following alcohol consumption, which could be related to brain damage caused by a local accumulation of acetaldehyde. Moreover, the involvement of ADH in the synthesis of retinoic acid suggests a role for the enzyme in the regulation of adult brain functions. The impairment of retinol oxidation by competitive inhibition of ADH in the presence of ethanol may be an additional origin of CNS abnormalities caused by ethanol.

Can nootropic drugs be effective against the impact of ethanol teratogenicity on cognitive performance?

Rats exposed pre- (PA) and postnatally (PNA) to ethanol at a dose of 1 g/kg for 24 h developed fetal alcohol effects (FAE). This was measured using a condition-reflex method for active avoidance with punishment reinforcement (shuttle-box) in which pronounced learning and memory deficits in 3-month-old rats were found after ethanol exposure (Vaglenova and Petkov, 1998. Fetal alcohol effects in rats exposed pre- and postnatally to a low dose of ethanol. Alcohol. Clin. Exp. Res. 22(3), 697--703). In the present study the effects of piracetam (Pyramem) at a dose of 600 mg/kg body weight, aniracetam at 50 mg/kg, and meclophenoxate (Centrophenoxine) at 100 mg/kg were studied. The drugs were administered orally during 10 days to separate groups of naive and pre- and postnatally exposed to ethanol rats. All the investigated nootropic drugs showed a significant possibility to alleviate learning and memory disability of rats with FAE. Aniracetam was administered to 1-month-old rats, demonstrating a prolonged (2 months) therapeutic effect, observed in rats aged 3 months. As previously reported (Vaglenova and Petkov, 1998), between male rats with FAE and controls, 66 and 33% were 'poor learners', respectively. In all nootropic treatment groups the percentage of 'poor learners' dropped to 28%. The positive effects of piracetam, aniracetam and meclophenoxate suggest that these drugs could be used for both treatment and prophylactic of FAE-connected disturbances of cognition.

Memory effects of the Ca2+ and 5-HT antagonists dotarizine and flunarizine.

In experiments on Wistar and Long Evans rats, using behavioral methods for passive (step-down and step-through) and active (shuttle-box two-way avoidance with punishment reinforcement) the newly synthesized diphenyl-methyl-piperazine derivative with Ca2+ and 5-HT antagonistic action dotarizine (DOT) administered repeatedly at oral doses of 50 and 10 mg/kg in some cases improve memory process. Under the same experimental conditions the chemically related to dotarizine Ca2+ antagonist flunarizine significantly facilitated retention. In old (Long Evans and Wistar) rats DOT in large dose decreases values of learning criterion. Probably this is a manifestation of the inherent to drugs with nootropic action "therapeutic window". Earlier investigations of the same and other authors suggest the participation of serotonergic neurotransmission in the mechanism of the memory effects of the drug DOT.

Fetal alcohol effects in rats exposed pre- and postnatally to a low dose of ethanol.

Wistar rats were exposed pre- and/or postnatally to a low dose of ethanol (1 g/kg of body weight of dams/day) via maternal peroral intubation. This dose significantly increased the mortality rate (23 to 32% vs. 7% in controls) in offspring exposed to ethanol during pregnancy, with a continued postnatal exposure having no additional effect. However, offspring cross-fostered to dams that had been exposed to ethanol only during gestation (the offspring themselves never being directly exposed to ethanol) displayed an even greater (59%) mortality. Growth of the offspring was initially delayed, but 9 weeks after birth their body weight reached that of the controls. The two-way active avoidance test showed an impairment, compared with the controls, of learning and memory in both male and female adolescent (9-week-old) rats, as well as in male (but not in female) 5-month-old rats born of dams exposed to ethanol during gestation and lactation. In the group of male rats treated prenatally and postnatally with ethanol, 60% were "poor learners," compared with 33% in the control group. Results suggest that ethanol at a dose of 1 g/kg/day administered to dams during gestation and lactation produced growth and behavioral changes in the offspring.

Alcohol dehydrogenase of human and rat blood vessels. Role in ethanol metabolism.

Alcohol dehydrogenase (ADH) activity has been detected in all arteries and veins examined from humans and rat. In distinct human autopsy vessels, activity values range from 0.9 +/- 0.2 to 9.9 +/- 7.7 mU/mg. Distribution of the activity in human aorta was: intima (23.5%), media (74%) and adventia (2.5%). In most of the samples the beta1 beta1 isozyme of class I ADH was the only form responsible for the ADH activity. Class IV ADH (sigma sigma-ADH) was present in three of the 28 individuals examined. The rat blood vessels showed class IV, but not class I, ADH localized in endothelium and media. The physiological role of vascular ADH is probably related to retinoid metabolism and elimination of lipid peroxidation aldehydes. A contribution to human ethanol metabolism is supported by the significant amount of low-Km activity and the extension of the vascular system.

Behavioral effects of the Ca2+/5-HT antagonist dotarizine.

The diphenyl-methyl-piperazine derivatives with Ca(2+)-antagonistic effect dotarizine (DOT), Fl-6020 and flunarizine were investigated in experiments on rats. The substances tested were administered repeatedly at an oral dose of 50 mg/kg. Behavioral methods were used to study the exploratory activity when the animals were placed in an environment that was unfamiliar to them (the chamber of the Opto Varimex apparatus), the elevated plus-maze method for examining the effect on anxiety, and the method of recording changes in motor activity (using the Automex II apparatus). DOT was found to increase motor activity and to have an anxiolytic effect. Combination of DOT--a compound with Ca(2+)--and 5-HT2-receptor antagonistic action--and the 5-HT-receptor agonists and antagonists used (buspirone, NAN190, pindolol, ritanserin and ondansetron) resulted in such changes in the development of habituation and in anxiety, which suggest that the modulating effects of DOT depend but partly on its typical interaction with the 5-HT2 receptor. Apparently, the Ca(2+)-antagonistic action of DOT plays a definite role, changing its biological activity depending on the 5-HT receptor subtype at the level of which the interaction is taking place.

Effects of cytidine diphosphate choline on rats with memory deficits.

The effects of cytidine diphosphate choline (CDP-choline, CAS 987-78-0) on learning and memory in rats with memory deficits were examined using behavioral methods of active avoidance with punishment reinforcement (shuttle-box), passive avoidance with punishment reinforcement (step-through and step-down), and active avoidance with positive (alimentary) reinforcement (staircase-maze). In the majority of experiments CDP-choline was applied orally at doses of 10-50 or 100 mg/kg daily for 7 days before the training session. The experiments were carried out on young-adult (aged 5 months) and old (aged 22 months) rats and on rats with a low capability for retention of learned behavior. Memory deficits were induced by the muscarinic cholinoceptor antagonist scopolamine (in young and old rats and mice), by the alpha 2-adrenoceptor agonist clonidine, by electroconvulsive shock, and by hypoxy. Memory deficits were also induced in rats offspring of dams that had been exposed to alcohol during pregnancy and lactation. The results suggest that CDP-choline acts as a memory-enhancing drug and that its effect is particularly pronounced in animals with memory deficits.

Learning and memory in rats exposed pre- and postnatally to alcohol. An attempt at pharmacological control.

Using conditioned-reflex methods for active and passive avoidance with punishment reinforcement, we found pronounced memory deficits in 12-week old rats exposed perinatally to alcohol (FAS rats). Impairment of memory was observed not only with the high dose of 9 g ethanol/kg body weight (ingested with tap water in a 6% solution) to which dams were exposed during pregnancy and lactation, but also with the ten-fold lower dose of 1 g ethanol/kg body weight (0.6% ethanol). The nootropic drugs citicholine, piracetam and meclofenoxate administered orally for five days before the training session were effective in decreasing memory deficits; particularly pronounced was the effect of piracetam and meclofenoxate. The benzodiazepine tranquilizer diazepam additionally impaired learning and memory in FAS rats. It is suggested that nootropics could be used to decrease the cognitive disturbances in some humans born to alcoholic mothers.

Changes in the synapses of rat hippocampus following pre- and postnatal alcohol exposure.

Quantitative and qualitative changes in the synapses in stratum lacunosum-moleculare of rat hippocampus following pre- and postnatal alcohol exposure were studied. Dense and lamellar bodies, damaged mitochondria, autophagic vacuoles and dilatated cisterns of the smooth endoplasmic reticulum were seen in axons and dendrites. The enlarged glial processes were also found. The area and the vesicle number of presynaptic terminals were decreased in CA1 and CA3 hippocampal fields, while the vesicle number per area of synaptic contact zones (SCZ) was decreased only in field CA3. The relative and absolute lengths of the SCZ, the total length and total surface of the SCZ were quite differently changed in both fields. The synaptic density was insignificantly increased. The synaptic changes are thought to be due to the impaired development of the pyramidal cell dendrites in the hippocampus and their afferents.

Effects of ethanol administered during pregnancy and lactation on the offspring survival, growth and exploratory behavior.

The survival (mortality rate), growth (body weight) and exploratory behavior (open field test) of the offspring of Wistar rats, treated with ethanol, were studied. Ethanol at a dose of 9 g/kg/day was applied as 6% aqueous solution (v/v). One group of rats received ethanol (E) during pregnancy and lactation (E + E), in another group E was substituted by water (W) during lactation (E + W) and a third group of rats received E only during lactation (W + E). The mortality rate in the offspring of the E + E and E + W groups of dams was significantly increased. The body growth of the offspring of the E + E group was significantly delayed as compared to controls. Inhibited exploratory activity, impaired habituation capacity and disturbed emotional balance were found in the 9-week old offspring of the E + W, W + E and E + E groups of dams respectively. It is suggested that pre- and/or postnatal ethanol received even at low concentrations with drinking water leads to long-lasting CNS disturbances characteristic of the fetal alcohol syndrome (FAS).

Enzyme histochemical studies on rat amygdala in fetal alcohol syndrome.

Histochemical studies were made of the activity of oxidative and hydrolytic enzymes in rat amygdala in FAS (Foetal Alcohol Syndrome). Ethanol in a dose of 9 g/kg/day was administered to rats during pregnancy and lactation in 6% aqueous solution as the only available liquid. The control rats received an equivalent diet in which ethanol was substituted by an equicaloric amount of sucrose. The offsprings were examined at the end of the 6th postnatal week. The activity of the lysosome and membrane enzymes, as well of some enzymes participating in the neurotransmission, was changed. A different decrease of the activity of oxidoreductases of Krebs cycle, glycolysis and pentose cycle was observed. The changes in the enzyme activity in the amygdala in FAS suggest alterations in the metabolism of the nervous tissue, rather than structural damages of cell organelles.

Differences between the effects of two meclofenoxate doses on active training for two-way avoidance in rats.

Male Wistar rats were trained for active two-way avoidance in a shuttle-box apparatus for five consecutive days. Meclofenoxate, in doses of 100 and 300 mg/kg i.p., was applied in two separate groups of rats, 60 min before the beginning of the training. Meclofenoxate (Mf), applied in a dose of 100 mg/kg, slightly improved the avoidance training, whereas in a dose of 300 mg/kg it "deteriorated" learning during the last two training days. Retrospective analysis of the experimental results showed that Mf caused a dose-dependent increase in the percentage of rats who were "poor learners", compared with the percentage of "poor learners" among the controls. On the other hand, the Mf-treated "good learners" manifested an essentially better capacity for avoidance training, compared with the control "good learners". The data obtained suggest that Mf increases the adaptational capacities of rats trained for active avoidance possibly in two different ways: in part of the animals it causes improvement of the conditioned reflex activity, in another part of the animals it increases their resistance to the stressogenic stimuli used during such a training.