Long-lasting tolerance to stimulatory effects of perinatal caffeine treatment.

Pregnant rats were treated with caffeine in their drinking water at doses of 136.3 mg/kg/day during gestation and 222.2 mg/kg/day during lactation. The resulting offspring at 60 days of age (40 days after drug withdrawal) were tested in an activity monitor cage. Spontaneous locomotor activity and that induced by caffeine (10, 30, 60 mg/kg/day) were observed. Treated rats showed apparent tolerance to caffeine-induced motility. Therefore perinatal caffeine treatment seems to induce long-lasting tolerance. This finding contrasts with those previously reported for chronic caffeine treatment in adult rats, where tolerance disappears after only 2-3 weeks following drug withdrawal.

Early postnatal chlordiazepoxide administration: permanent behavioural effects in the mature rat and possible involvement of the GABA-benzodiazepine system.

The long term behavioural and biochemical effects of chronic chlordiazepoxide treatment during the period of neuronal maturation in the rat have been investigated. The administration to lactating mothers of chlordiazepoxide at very low doses (0.22 and 2.6 mg/kg) in their drinking water affects both behavioural and biochemical parameters in offspring at 60 days of age and undrugged since weaning. A deficit in the acquisition of the conditioned avoidance response in treated rats was observed, although no significant difference in spontaneous locomotor activity between control and treated rats was found. 3H-Flunitrazepam binding sites in cerebral cortex and hippocampus were decreased by the treatment, whereas no change was detected in cerebellum. Moreover, 3H-muscimol binding sites increased in hippocampus with no changes in cerebral cortex and cerebellum. According to the different regional distribution of benzodiazepine type 1 and type 2 receptors, we suggest that type 2 receptors are selectively affected by the treatment, and that the GABAergic receptor system is also permanently altered by administration of chlordiazepoxide during early postnatal life.

Loss of intrinsic striatal neurons after methylazoxymethanol acetate treatment in pregnant rats.

Treatment of pregnant rats with methylazoxymethanol (MAM) induces a marked microencephaly in the offspring. The striatum is one of the brain areas affected by treatment. We show that in striatum there is a 30% loss of dopamine (DA)-dependent adenylate-cyclase activity: this indicates that cells bearing type 1 DA receptors are affected by MAM treatment. Moreover, since kainic acid retains its neurotoxic activity, corticostriatal fibers do not seem to be affected, despite the dramatic reduction of cortex size.

Morphometrical and microdensitometrical studies on peptide- and tyrosine hydroxylase-like immunoreactivities in the forebrain of rats prenatally exposed to methylazoxymethanol acetate.

Methylazoxymethanol acetate (MAM Ac) injected into pregnant rats at a dose of 25 mg/kg at gestational day 15 causes microcephaly due to an atrophy of various telencephalic areas, mainly neocortex, hippocampus and basal ganglia. Previous studies demonstrated alterations in various neurochemical markers of classical transmitter systems in these regions. The present paper deals with changes in peptide and tyrosine hydroxylase (TH)-containing neurons in MAM Ac-induced microcephaly using immunocytochemistry coupled with computer-assisted morphometry and microdensitometry. No change in the number of vasoactive intestinal polypeptide (VIP)-immunoreactive neurons in the neocortex and neuropeptide Y (NPY)-immunoreactive neurons in the nucleus caudatus-putamen was found whereas cholecystokinin (CCK)-and NPY-immunoreactive neurons in the neocortex and CCK- and VIP-immunoreactive neurons in the hippocampus were decreased. The reduction of the latter peptide containing neuronal populations led to a maintained density of cells in MAM Ac-exposed rats, due to the parallel reduction of the overall mass of these regions. TH immunoreactivity was found to be unchanged in the basal ganglia, and increased in the cerebral cortex in agreement with previous reports on noradrenaline cortical system after MAM Ac exposure. The present results show a heterogenous vulnerability of different peptide immunoreactive neuronal populations to MAM Ac exposure. The sparing of VIP- and NPY-immunoreactive neurons may be due to their late development in the neocortex and striatum, respectively. The hypothesis is introduced that cortical VIP interneurons can develop independent of marked alterations in the intrinsic circuitry of the cortical region.

Treatment with methylazoxymethanol at different gestational days: physical, reflex development and spontaneous activity in the offspring.

Pregnant rats were injected with a single dose of methylazoxymethanol (MAM, 25 mg/kg) on gestational day 14, 15, 16, 17, 18 or 19 and offspring were tested for their physical development, reflex development and spontaneous activity. MAM treatment did not affect gestational and litter parameters at any of the time of administration studied. Treatment at gestational day 14 (GD14) had the most severe effect on functional neurodevelopment until weaning: righting reflex at surface, chimney test, horizontal wire test resulted altered. Administration at GD15, 16, 18, 19 did not affect the performance in these tests. Offspring treated at GD17 showed a delayed eye opening and an impaired performance in the horizontal wire test. When tested at 50 days of age on the rotarod, all the treated groups performed worse than controls with the exception of GD19 treated offspring. Administration at GD14 and GD15 resulted in increased spontaneous activity of the offspring at 21 days but not at 60 days of age. Different degrees of microencephaly were observed for all treated groups. The results indicate that alterations of physical and behavioral development induced by MAM treatment are dependent on the time of MAM administration, and specific behavioral tests are able to detect different abnormalities and differentiate among treatment groups. Some alterations observed in MAM rats undergo to adaptive changes during maturation of the CNS.

Treatment with methylazoxymethanol at different gestational days: two-way shuttle box avoidance and residential maze activity in rat offspring.

Pregnant rats were injected with a single dose of methylazoxymethanol (MAM, 25 mg/kg) on gestational days 14, 15, 16, 17, 18 or 19 which resulted in various degrees of microencephaly. Offspring were tested on a two-way shuttle box avoidance and residential maze activity at 60-90 days of age. Rats treated on gestational day 19 (GD19) were severely impaired in the acquisition of the two-way shuttle box task whereas the other groups did not show any significant difference from controls. Spontaneous activity measured for 23 hr in the residential maze was altered as total, time-course and pattern depending on the time of MAM administration: treatment on GD14 prolonged exploratory behavior, treatment on GD15 and GD16 increased nocturnal activity, treatment on GD16 and GD17 induced changes in locomotion patterns and treatment on GD18 and GD19 decreased total activity. These findings indicate that treatment with MAM results in selective deficits in the acquisition of a shuttle box avoidance and alterations of locomotion patterns in the offspring which are dependent on the time of administration.

Microencephalic rats as a model for cognitive disorders.

The administration of the antimitotic compound methylazoxymethanol (MAM) to gestating rats induces a dose-dependent atrophy of specific brain areas in the offspring. This specificity is strictly dependent upon the time of MAM administration. When given at day 15 of gestation only the cortex, hippocampus, and striatum are affected, whereas when given to rat pups at postnatal day 1, the atrophy is apparent only in the cerebellum. The microencephalic offspring of dams treated at day 15 of gestation develop normally to adulthood, without manifest signs of this profound telencephalic contraction. Behavioral abnormalities are observable when subjecting these animals to tests that involve learning. The deficit in associative behavior might have its anatomical and neurochemical counterpart in the disruption of the neuronal circuitry in the neocortex, where about 50% of interneurons are absent in layers II-IV after a dose of MAM of 25 mg/kg. Loss of intrinsic neurons occurs also in the striatum, as revealed by neurochemical and pharmacological analysis. Indeed, MAM rats show a reduced dopamine-dependent adenylate cyclase activity and a reduced motor stimulation in response to dopaminergic stimulants. MAM rats are therefore an interesting animal model of chronic brain damage induced transplacentally, which could serve for studying adaptive mechanisms of the CNS to this damage and its pharmacological manipulation.

Nocturnal hyperactivity induced by prenatal methylazoxymethanol administration as measured in a computerized residential maze.

Treatment of female Sprague-Dawley rats with 15 or 25 mg/kg (IP) of methylazoxymethanol acetate (MAM) at gestational day 15 (15 DG) resulted in a dose-dependent reduction of total brain weight of the adult offspring. When tested for spontaneous activity in a residential maze over a 23 hour period, those animals treated with the highest dose of MAM showed an increase in both locomotion and local activity during night hours without changes in the structure of behavior. Animals treated with 15 mg/kg of MAM showed no difference in activity compared to controls despite a significant reduction in brain weight.

Studies on the development and stability of resistance of Helicobacter pylori to metronidazole and clarithromycin.

Thirty-one strains of Helicobacter pylori, susceptible to metronidazole and clarithromycin, were isolated from 31 biopsy samples from patients with various gastric pathologies. We investigated the possibility of selecting resistant strains and the stability of resistance by exposing isolates to increasing subinhibitory concentrations of metronidazole and clarithromycin using a serial passages technique. Resistance to metronidazole was obtained in 100% of the isolates, while 32.2% displayed resistance to clarithromycin. Resistance to metronidazole was stable in 93.5% (29 of 31) of the isolates, whereas 100% (10 of 10) of the strains were stably resistant to clarithromycin. The stability of the resistance that occurred after three passages on medium containing the two drugs was statistically significant (P <0.001). Thus, the technique of serial passages in vitro could be useful as a first screening in selecting drugs for use in therapeutic protocols for clinical trials.

Cholinergic hyperinnervation in the cerebral cortex of microencephalic rats does not result in muscarinic receptor down-regulation or in alteration of receptor-stimulated phosphoinositide metabolism.

Administration of methylazoxymethanol (MAM; 25 mg/kg) to pregnant rats at gestational day 15 (GD 15) induces a marked reduction of telencephalic areas of the offspring brain. Previous neurochemical studies demonstrated a marked cholinergic hyperinnervation in the cerebral cortex of microencephalic rats. In this study we have evaluated whether this cholinergic hyperinnervation could result in altered functionality of muscarinic receptors. Acetylcholinesterase activity (AChE) was increased by 69% in the cerebral cortex of MAM treated rats, confirming a relative hyperinnervation, whereas in the hippocampus and striatum no significant changes were observed. Despite the marked hyperinnervation, in the cerebral cortex of microencephalic rats neither muscarinic receptor-stimulated phosphoinositide metabolism nor muscarinic receptor density were altered. No differences in receptor density were also observed in the hippocampus and striatum. Chronic diisopropylfluorophosphate (DFP) administration induced a marked decrease of AChE activity and down-regulation of muscarinic receptors whereas atropine administration resulted in receptor up-regulation in cerebral cortex, striatum and hippocampus of both control and MAM rats. The results confirm a relative cholinergic hyperinnervation in the cerebral cortex of microencephalic rats and demonstrate that the regulation of muscarinic receptor-stimulated phosphoinositide metabolism and muscarinic receptor plasticity is not modified in a condition of increased cholinergic presynaptic terminals.

Effect of prenatal treatment with methylazoxymethanol on carbachol-, norepinephrine- and glutamate-stimulated phosphoinositide metabolism in the neonatal, young, and adult offspring.

Carbachol-, norepinephrine- and glutamate-stimulated phosphoinositide metabolism was investigated in the neonatal, young and adult cerebral cortex slices of rats prenatally treated with methylazoxymethanol (MAM) on gestational day 15 (GD15) or GD19. In rat offspring treated on GD15 there was a significant reduction in the accumulation of [3H]inositol phosphates induced by carbachol and a significant increase in the accumulation of [3H]inositol phosphates induced by norepinephrine on day 7, whereas no changes were observed at the other ages. No significant changes, on the other hand, were observed for glutamate-stimulated phosphoinositide metabolism in GD15 treated rats and for carbachol-, norepinephrine- and glutamate-stimulated phosphoinositide metabolism in animals treated on GD19 at any of the different ages evaluated. These results indicate that treatment with MAM on GD15, which results in a marked microencephaly, causes a marked alteration of muscarinic and alpha 1-adrenergic receptor-stimulated phosphoinositide metabolism during brain development and that these alterations undergo adaptive changes in the adult brain.

7-Substituted theophyllines. Part I. Nicotinamide derivatives.

Some nicotinamides derived from 7-substituted theophyllines were prepared and pharmacologically screened. They showed a very low coronarodilatory activity, a remarkable antispastic activity and a low toxicity.

Perinatal caffeine treatment: behavioral and biochemical effects in rats before weaning.

Administration of drinking water containing 0, 0.02%, 0.04% and 0.08% of caffeine to female rats throughout gestation and lactation affects several behavioral parameters in the offspring. Righting reflexes, swimming ability development, motor coordination and muscle tone were affected. The activity of these animals, as measured with an open-field test at weaning (i.e., at the end of the treatment), was reduced. The effects observed were dose-dependent. However, for righting reflexes the dose-dependency was direct (the highest dose producing maximal effects), whereas in all the other tests, the dose-dependency was inverse, the lowest dose producing maximal effects and the highest dose producing no effects. This might reflect the presence of subclasses of receptors having different affinities for adenosine, mediating opposite effects and antagonized by caffeine. On the other hand, perinatal caffeine effects are certainly not mediated by blockade of phosphodiesterases, since cAMP levels at the end of the treatment were dose-dependently reduced. This study shows therefore that administration of caffeine to rat dams is able to influence the neurobehavioral development of the offspring. Moreover, all the doses utilized and corresponding to 27, 58 and 108 mg/kg, were able to produce all or some of the mentioned effects, indicating that further testing with doses lower than 27 mg/kg is required to find a dose which does not affect the offspring.