methylazoxymethanol and Abnormalities--Drug-Induced

In the last decade, the recognition of the high frequency of cortical malformations among patients with epilepsy especially children, has led to a renewed interest in the study of the pathophysiology of cortical development. This field has also been spurred by the recent development of several experimental genetic and non-genetic, primarily rodent, models of cortical malformations. Epileptiform activity in these animals can appear as spontaneous seizure activity in vivo, in vitro hyperexcitability, or reduced seizure susceptibility in vitro and in vivo. In the neonatal freeze lesion model, that mimics human microgyria, hyperexcitability is caused by a reorganization of the network in the borders of the malformation. In the prenatal methylazoxymethanol model, that causes a diffuse cortical malformation, hyperexcitability is associated with alteration of firing properties of discrete neuronal subpopulations together with the formation of bridges between normally unconnected structures. In agreement with clinical evidence, these experimental data suggest that cortical malformations can both form epileptogenic foci and alter brain development in a manner that causes a diffuse hyperexcitability of the cortical network.

Topics: Abnormalities, Drug-Induced; Animals; Cerebral Cortex; Disease Models, Animal; Epilepsy; Hippocampus; Humans; Methylazoxymethanol Acetate; Mice; Mice, Knockout; Mitosis; Mutation; Nervous System Malformations; Neural Conduction; Neural Pathways; Rats; Rats, Mutant Strains; Teratogens

One of the most common causes of neurological disabilities are malformations of cortical development (MCD). A useful animal model of MCD consists of prenatal exposure to methylazoxymethanol (MAM), resulting in a postnatal phenotype characterized by cytological aberrations reminiscent of human MCD. Although postnatal effects of MAM are likely a consequence of prenatal events, little is known on how the developing brain reacts to MAM. General assumption is the effects of prenatally administered MAM are short lived (24 h) and neuroblast-specific. MAM persisted for several days after exposure in utero in both maternal serum and fetal brain, but at levels lower than predicted by a neurotoxic action. MAM levels and time course were consistent with a different mechanism of indirect neuronal toxicity. The most prominent acute effects of MAM were cortical swelling associated with mild cortical disorganization and neurodegeneration occurring in absence of massive neuronal cell death. Delayed or aborted vasculogenesis was demonstrated by MAM's ability to hinder vessel formation. In vitro, MAM reduced synthesis and release of VEGF by endothelial cells. Decreased expression of VEGF, AQP1, and lectin-B was consistent with a vascular target in prenatal brain. The effects of MAM on cerebral blood vessels persisted postnatally, as indicated by capillary hypodensity in heterotopic areas of adult rat brain. In conclusion, these results show that MAM does not act only as a neurotoxin per se, but may additionally cause a short-lived toxic effect secondary to cerebrovascular dysfunction, possibly due to a direct anti-angiogenic effect of MAM itself.

Topics: Abnormalities, Drug-Induced; Animals; Aquaporin 1; Brain; Cerebral Arteries; Cerebral Cortex; Disease Models, Animal; Female; Methylazoxymethanol Acetate; Neovascularization, Physiologic; Nervous System Malformations; Neurotoxins; Pokeweed Mitogens; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Rats, Sprague-Dawley; Teratogens; Vascular Endothelial Growth Factor A

We investigated the cognitive consequences of a prenatal injection of the mitotic inhibitor methylazoxymethanol (MAM) into pregnant rats at embryonic day 15 (E15) or 17 (E17). The male offspring were tested when adult on a version of the radial-arm maze task that assesses spatial working memory with an extended delay, where performance is dependent, in part, on the hippocampal-prefrontal circuit. A major impairment of spatial learning was observed in E15 MAM rats. However, the E17 MAM rats did learn the rule but were impaired selectively in the 30-min delay-interposed task. Morphologically, the E15 MAM rats exhibited dramatic gross brain abnormalities, whereas the E17 MAM animals displayed aberrant cell migration in the hippocampus and a disrupted laminar pattern in the neocortex. These results suggest that late gestational MAM injection (E17) causes a cognitive impairment in a prefrontal cortex-hippocampus-dependent working memory task. This approach could provide a new developmental model of disorders associated with working memory deficits, such as schizophrenia.

Topics: Abnormalities, Drug-Induced; Animals; Disease Models, Animal; Drug Administration Schedule; Female; Hippocampus; Male; Maze Learning; Memory Disorders; Methylazoxymethanol Acetate; Organ Size; Prefrontal Cortex; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Rats, Sprague-Dawley; Time Factors

Treatment of pregnant ferrets on embryonic day 24 (E24) with the antimitotic methylazoxy methanol (MAM) leads to a specific constellation of effects in newborn kits, which include a very thin and poorly laminated neocortex, disruption of radial glial cell morphology with early differentiation into astrocytes, and abnormal positioning of Cajal-Retzius cells. We suggest that MAM treatment on E24 results in this model of cortical dysplasia by eliminating a population of cells that produce a factor capable of maintaining radial glia in their normal morphology. The abnormal radial glia, either alone or in combination with other abnormal features, are likely to prevent proper migration into the cortical plate. To test the possibility that normal cortex can provide the missing substance that influences radial glia, slices of E24 MAM-treated cortex were removed at postnatal day 0 (P0) and cultured adjacent to explants of P0 normal cortical plate. By labelling a small number of cells with injections of fluorescent dextrans into the cultured slices, we found that abnormal radial glia in MAM treated slices cocultured adjacent to normal cortical plate were restored toward normal, in comparison to E24 MAM treated slices cultured alone and in other control conditions. We also found that abnormally positioned Cajal-Retzius cells move into the marginal zone and that neurons are able to migrate into the cortical plate more effectively in the coculture condition. These data indicate that normal cortical plate of ferrets contains a factor causing radial glia to maintain their elongated morphology; the improved position of radial glia encourages repositioning of Cajal-Retzius cells and improved neuronal migration into the cortical plate.

Topics: Abnormalities, Drug-Induced; Animals; Antimetabolites; Brain; Bromodeoxyuridine; Cell Count; Cell Movement; Cerebral Cortex; Ciliary Neurotrophic Factor; Coculture Techniques; Female; Ferrets; Fluorescent Dyes; Image Processing, Computer-Assisted; Methylazoxymethanol Acetate; Models, Neurological; Neuroglia; Organ Culture Techniques; Pregnancy; Teratogens

Neuronal migration disorders (NMDs) can be associated with neurological dysfunction such as mental retardation, and clusters of disorganized cells (heterotopias) often act as seizure foci in medically intractable partial epilepsies. Methylazoxymethanol (MAM) treatment of pregnant rats results in neuronal heterotopias in offspring, especially in hippocampal area CA1. Although the neurons in dysplastic areas in this model are frequently hyperexcitable, the precise mechanisms controlling excitability remain unclear. Here, we used IR-DIC videomicroscopy and whole cell voltage-clamp techniques to test whether the potent anti-excitatory actions of neuropeptide Y (NPY) affected synaptic excitation of heterotopic neurons. We also compared several synaptic and intrinsic properties of heterotopic, layer 2-3 cortical, and CA1 pyramidal neurons, to further characterize heterotopic cells. NPY powerfully inhibited synaptic excitation onto normal and normotopic CA1 cells but was nearly ineffective on responses evoked in heterotopic cells from stimulation sites within the heterotopia. Glutamatergic synaptic responses on heterotopic cells exhibited a comparatively small, D-2-amino-5-phosphopentanoic acid-sensitive, N-methyl-D-aspartate component. Heterotopic neurons also differed from normal CA1 cells in postsynaptic membrane currents, possessing a prominent inwardly rectifying K(+) current sensitive to Cs(+) and Ba(2+), similar to neocortical layer 2-3 pyramidal cells. CA1 cells instead had a prominent Cs(+)- and 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride-sensitive I(h) and negligible inward rectification, unlike heterotopic cells. Thus heterotopic CA1 cells appear to share numerous physiological similarities with neocortical neurons. The lack of NPY's effects on intra-heterotopic inputs, the small contribution of I(h), and abnormal glutamate receptor function, may all contribute to the lowered threshold for epileptiform activity observed in hippocampal heterotopias and could be important factors in epilepsies associated with NMDs.

Topics: Abnormalities, Drug-Induced; Animals; Electrophysiology; Epilepsy; Excitatory Postsynaptic Potentials; Female; Hippocampus; Histocytochemistry; Membrane Potentials; Methylazoxymethanol Acetate; Neocortex; Neuropeptide Y; Potassium Channel Blockers; Potassium Channels; Pregnancy; Pyramidal Cells; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Synaptic Membranes; Teratogens

A double methylazoxymethanol (MAM) intraperitoneal injection was prenatally administered to pregnant rats at gestational day 15 to induce developmental brain dysgeneses. Thirty adult rats from 8 different progenies were investigated with a combined electrophysiological and neuroanatomical analysis. The offspring of treated dams was characterized by extensive cortical layering abnormalities, subpial bands of heterotopic neurons in layer I, and subcortical nodules of heterotopic neurons extending from the periventricular region to the hippocampus and neocortex. The phenotype of cell subpopulations within the heterotopic structures was analyzed by means of antibodies raised against glial and neuronal markers, calcium binding proteins, GABA, and AMPA glutamate receptors. Neurons within the subcortical heterotopic nodules were characterized by abnormal firing properties, with sustained repetitive bursts of action potentials. The subcortical nodules were surrounded by cell clusters with ultrastructural features of young migrating neurons. The immunocytochemical data suggested, moreover, that the subcortical heterotopia were formed by neurons originally committed to the neocortex and characterized by morphological features similar to those found in human periventricular nodular heterotopia. The present study demonstrates that double MAM treatment at gestational day 15 induces in rats developmental brain abnormalities whose anatomical and physiological features bear resemblance to those observed in human brain dysgeneses associated with intractable epilepsy. Therefore, MAM treated rats could be considered as useful tools in investigating the pathogenic mechanisms involved in human developmental brain dysgeneses.

Topics: Abnormalities, Drug-Induced; Animals; Brain; Cerebral Ventricles; Choristoma; Embryonic and Fetal Development; Female; Hippocampus; Humans; Immunohistochemistry; Injections, Intraperitoneal; Methylazoxymethanol Acetate; Microscopy, Electron; Neurons; Neurotoxins; Pregnancy; Rats; Rats, Sprague-Dawley

Postnatal changes in the fiber distribution and release of 5-HT in the somatosensory cortex (Sm) of methylazoxymethanol acetate (MAM)-induced microencephalic rats were studied. A transient accumulation of serotonergic fibers was observed in the Sm of the control and MAM rats in the first 2 weeks following birth. However, the density of serotonergic fibers was higher in the MAM rats than in the controls, and the distribution pattern of serotonergic fibers was more extensive in the MAM rats. The microdialysis indicated a high basal level and a delayed K+ -evoked 5-HT release in the Sm of MAM rats on postnatal day 10. The present results suggest morphological and functional alterations of serotonergic fibers in the Sm of MAM rats during the critical period of cortical formation.

Topics: Abnormalities, Drug-Induced; Aging; Animals; Female; Immunohistochemistry; Male; Methylazoxymethanol Acetate; Microcephaly; Microdialysis; Rats; Rats, Sprague-Dawley; Serotonin; Somatosensory Cortex; Teratogens

Prenatal treatment with methylazoxymethanol (MAM) in rats generates animals with a diffuse cortical malformation associated with hyperexcitability. These alterations are reminiscent of the cortical malformations associated with epilepsy in children. We hypothesised that one of the mechanisms supporting hyperexcitability in MAM rats could be the presence of abnormal cortical connections in the malformed cortex. Using a variety of anatomical techniques, we provide evidences for three types of such abnormal connections: (i) tangential bundles of corticocortical fibres in and below the neocortical molecular layer; (ii) partial deafferentation of neocortical heterotopias by afferent cortical fibres whatever their location; (iii) exuberant innervation of hippocampal CA3 pyramidal cells by mossy fibres that form ectopic mossy boutons on their basal dendrites. We conclude that these abnormal intrinsic cortical connections may support the propagation of paroxymal activity in the neocortex of MAM-treated rats.

Topics: Abnormalities, Drug-Induced; Animals; Cerebral Cortex; Embryo, Mammalian; Female; Hippocampus; Methylazoxymethanol Acetate; Neocortex; Neural Pathways; Pregnancy; Rats; Rats, Wistar; Teratogens

Cerebellar pattern formation was investigated in rats treated with DNA modifying agents. Animals were subjected to combinations of daily injections of methylazoxymethanol acetate (MAM) for the last 6 days gestation and/or localised X-irradiation of the hindbrain on postnatal days 1 and 5 (P1 and P5). Animals were analysed on embryonic day 18 (E18), P0, P3, P7, and P14. Five parameters of the cerebellum were recorded from midsagittal sections: the number of primary lobules; the thickness of the external germinal layer (EGL); the density of cells in the internal granule cell layer (IGL) region; and the midsagittal area and perimeter. In addition, the laterolateral cerebellar distance was calculated. The data demonstrate that pre- and postnatal reduction of the EGL results in reduced cerebellar growth and folding. Cessation of the treatment at birth results in a recovery and eventual overproduction of EGL, but cerebellar growth and the development of fissures lags behind that of normal rats. Pre- and postnatal destruction of the EGL severely limited cerebellar growth and fissuration, and the cerebella contained only five primary lobules at P14. Rats subjected to postnatal X-irradiation alone had a similar low density of granule cells relative to those treated with a combination of prenatal MAM injections and postnatal X-irradiation, and yet the cerebella contained deeper fissures and more lobules (nine at P14). The data indicate that there are two phases of cerebellar folding: the establishment of five lobules that arise independent of granule cell production, and the granule cell-dependent expansion and partitioning of these five principal lobules during postnatal development. We propose that the lack of correlation between the severity of the granule cell loss and degree of lobulation in agranular rats indicates that granule cells exert an inductive influence over lobulation that is in part independent of the forces generated by their production and differentiation.

Topics: Abnormalities, Drug-Induced; Alkylating Agents; Animals; Cerebellar Diseases; Cerebellum; Female; Male; Methylazoxymethanol Acetate; Pregnancy; Prenatal Exposure Delayed Effects; Radiation Injuries, Experimental; Rats; Rats, Wistar

In humans, cortical malformations are highly epileptogenic. In rats, prenatal treatment with methylazoxymethanol (MAM) cause a diffuse cortical malformation that is yet not associated with seizures. We performed rapid hippocampal kindling in MAM and control rats. We show that MAM rats present (i) a lower initial afterdischarge threshold; (ii) a more rapid progression to generalized seizures. We conclude that MAM rats may serve as models for human epileptogenic cortical malformations.

Topics: Abnormalities, Drug-Induced; Animals; Cerebral Cortex; Electroencephalography; Female; Hippocampus; Kindling, Neurologic; Methylazoxymethanol Acetate; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Reaction Time; Seizures

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.

Topics: Abnormalities, Drug-Induced; Acetylcholinesterase; Animals; Atropine; Brain; Cerebral Cortex; Down-Regulation; Female; Isoflurophate; Maternal-Fetal Exchange; Methylazoxymethanol Acetate; Organ Size; Phosphatidylinositols; Pregnancy; Rats; Rats, Inbred Strains; Receptors, Muscarinic

Behavioral testings in methylazoxymethanol (MAM)-induced microcephalic rats were conducted. Pregnant Sprague-Dawley rats were treated intraperitoneally with 0, 20 or 40 mg/kg of MAM once a day on day 14 of gestation and were allowed to delivery. Male pups from each litter were examined for open field test at 6 weeks of age and shuttle-box avoidance test at 7 weeks or more of age. In the open field activity of pups, the counts of ambulation and locomoting distance in 40 mg/kg group have increased significantly as compared with those in control group. In the shuttle-box avoidance test, the avoidance response rate was dose-dependently high in the session of the 1st day. As to the interaction between the avoidance response rate and sequence of sessions, however, the avoidance response rate in 40 mg/kg group was significantly low. Rate of the rats with errors and number of response during the intertrial interval was significantly high in 40 mg/kg group. Thus, we could demonstrate functional disturbance in the memory retaining ability in utero MAM-exposed rats.

Topics: Abnormalities, Drug-Induced; Animals; Avoidance Learning; Dose-Response Relationship, Drug; Female; Male; Memory; Methylazoxymethanol Acetate; Microcephaly; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Rats, Sprague-Dawley

Methylazoxymethanol (MAM)-induced cerebral hypoplasia resulted in a significant increase in densities of both serotonin uptake sites in frontal cortex and dopamine uptake sites in striatum, suggesting serotonergic and dopaminergic axons terminals were compressed in the smaller brain volumes. The density of S2 serotonin receptors in MAM-lesioned frontal cortex was decreased probably due to down-regulation, while densities of D1 and D2 dopamine receptors in striatum were identical between MAM-lesioned rats and control rats.

Topics: Abnormalities, Drug-Induced; Animals; Biological Transport; Brain; Cerebral Cortex; Corpus Striatum; Dopamine; Female; Kinetics; Methylazoxymethanol Acetate; Pregnancy; Rats; Rats, Inbred Strains; Receptors, Serotonin; Reference Values; Serotonin; Teratogens

Prenatal treatment of rats on gestation day 15 with methylazoxymethanol (MAM) caused forebrain microencephaly. The behavioral analyses included measures of spontaneous motor activity and tests for cognitive deficits, and were performed when the rats had reached adult age. Female MAM-treated rats failed to demonstrate contextual control of latent inhibition, which confirms earlier findings with male rats. Male MAM-treated rats demonstrated a notable impairment of place navigation in a swim-maze, but showed as strong sensory preconditioning as the control animals. Biochemical analyses indicated considerable increases in catecholamine levels in the cerebral cortex, hippocampus and striatum. The cognitive deficits, characterised by the various conditioning (taste-aversion) and instrumental learning (swim-maze) tasks, suggested that the MAM rats are deficient in their capacity to attend selectively to the relevant stimulus in complex arrangements of the stimulus situation.

Topics: Abnormalities, Drug-Induced; Animals; Attention; Azo Compounds; Brain; Cognition Disorders; Female; Inhibition, Psychological; Learning Disabilities; Methylazoxymethanol Acetate; Pregnancy; Prenatal Exposure Delayed Effects; Rats; Rats, Inbred Strains; Reaction Time; Space Perception

The regional levels of several cell marker proteins in the brain and the ability of operant discrimination learning on a multiple fixed ratio (FR), fixed interval (FI) schedule were determined in rats with microencephaly induced by prenatal treatment with methylazoxymethanol (MAM), an antimitotic agent, on the 11 th to 13 th days (Group A) or on the 15 th day (Group B) of gestation. The cell marker proteins were determined with a sensitive enzyme immunoassay. Neuron-specific enolase (NSE; gamma gamma-enolase) had a significantly lowered level in the neocortex anterior in Group A. Non-neuronal enolase (NNE; alpha alpha-enolase) was significantly reduced in the superior colliculus, lateral geniculate body and optic nerve, but increased 1.5 fold in the retina in Group A. S-100b protein, a marker of astroglial cells, showed no significant change. As for the learning performance, the Group B animals showed an elevated behavioral activity and made evident discrimination between the FI and FR schedule. But Group A animals had prolonged FR components requiring responses to light on, and their spontaneous activity counts recorded by Automex showed an inhibition of behavior in light environments. These findings suggest a causative role of some developmental abnormality in the central visual system, indicated by the aberrant cell marker levels, in the disturbed learning ability of the Group A animals.

Topics: Abnormalities, Drug-Induced; Alkylating Agents; Animals; Azo Compounds; Brain; Brain Chemistry; Conditioning, Operant; Discrimination Learning; Female; Gestational Age; Locomotion; Methylazoxymethanol Acetate; Microcephaly; Organ Size; Phosphopyruvate Hydratase; Pregnancy; Rats; Rats, Inbred Strains; S100 Proteins

The retinofugal projections of albino rats made micrencephalic by prenatal exposure to the cytotoxic teratogen methylazoxymethanol acetate (MAM Ac) have been examined. The only abnormality noted was an increased projection to the lateral posterior nucleus of the thalamus in rats exposed to MAM Ac on embryonic day 15. The relatively normal retinofugal projections were surprising in view of the extensive damage induced by prenatal exposure to this drug.

Topics: Abnormalities, Drug-Induced; Animals; Female; Geniculate Bodies; Methylazoxymethanol Acetate; Microcephaly; Pregnancy; Rats; Rats, Inbred Strains; Retina; Superior Colliculi; Thalamic Nuclei; Visual Pathways

Topics: Abnormalities, Drug-Induced; Analysis of Variance; Animals; Azo Compounds; Behavior, Animal; Body Weight; Brain; Carcinogens; Female; Fetus; Learning; Male; Methylazoxymethanol Acetate; Motor Activity; Organ Size; Pregnancy; Rats; Rats, Inbred Strains; Reproduction