Reduced risk aversion and impaired short-term memory in juvenile rats with malformation of cortical development.

Malformation of cortical developments (MCDs) is currently an incurable disease and is associated with significant neuropsychological problems, such as intellectual disability, epilepsy, and anxiety disorders from a young age. Development of a suitable animal model and pathophysiological study is therefore necessary to better understand and treat MCDs from being an incurable disease. The Y-maze, open field, and fear conditioning studies were performed at postnatal days 40-44 to validate the behavioral phenotypes of the existing rat model of MCD with prenatal methylazoxymethanol exposure at their developmental period. The study results show that juvenile rats with MCD spent significantly less time inside the novel arms in Y-maze and less time in the peripheral zones of the open field. Additionally, the rats with MCDs showed attenuated freezing behavior to sound and light cues as well as to context after fear conditioning. This comprehensive behavioral analysis of rats with MCDs at the juvenile period indicate a lack of spatial memory, decreased anxiety, and learning disability in these rats, which is compatible with the human behavioral phenotype of MCDs and can be used as the behavioral biomarkers for future translational research.

Diminished excitatory synaptic transmission correlates with impaired spatial working memory in neurodevelopmental rodent models of schizophrenia.

Neurodevelopmental abnormalities are associated with cognitive dysfunction in schizophrenia. In particular, deficits of working memory, are consistently observed in schizophrenia, reflecting prefrontal cortex (PFc) dysfunction. To elucidate the mechanism of such deficits in working memory, the pathophysiological properties of PFc neurons and synaptic transmission have been studied in several developmental models of schizophrenia. Given the pathogenetic heterogeneity of schizophrenia, comparison of PFc synaptic transmission between models of prenatal and postnatal defect would promote our understanding on the developmental components of the biological vulnerability to schizophrenia. In the present study, we investigated the excitatory synaptic transmission onto pyramidal cells localized in layer 5 of the medial PFc (mPFc) in two developmental models of schizophrenia: gestational methylazoxymethanol acetate (MAM) administration and post-weaning social isolation (SI). We found that both models exhibited defective spatial working memory, as indicated by lower spontaneous alternations in a Y-maze paradigm. The recordings from pyramidal neurons in both models exhibited decreased spontaneous excitatory postsynaptic current (sEPSC), representing the reduction of excitatory synaptic transmission in the mPFc. Interestingly, a positive correlation between the impaired spontaneous alternation behavior and the decreased excitatory synaptic transmission of pyramidal neurons was found in both models. These findings suggest that diminished excitatory neurotransmission in the mPFc could be a common pathophysiology regardless of the prenatal and postnatal pathogenesis in developmental models of schizophrenia, and that it might underlie the mechanism of defective working memory in those models.

Memory deterioration based on the tobacco smoke exposure and methylazoxymethanol acetate administration vs. aripiprazole, olanzapine and enrichment environment conditions.

Enrichment environment conditions, as well as tobacco smoke exposure, may affect cognitive function (e.g. spatial memory) in an animal model of schizophrenia and schizophrenic patients. The aim of this study was to find whether spatial memory function impairment is found in methylazoxymethanol acetate treated rats (an animal model of schizophrenia) and whether aripiprazole (1.5 mg/kg) and olanzapine (0.5 mg/kg) modify these functions. We also were able to determine whether tobacco smoke exposure and enrichment environment conditions have an impact on drug efficacy. The effect of methylazoxymethanol acetate, tobacco smoke exposure, enrichment environment and the use of drugs were studied in the Morris Water Maze test (spatial memory). The results of our study clearly show that enriched environment may have a procognitive effect while tobacco smoke and methylazoxymethanol acetate have a contradictory effect. This paper also confirmed that the use of neuroleptics, namely ARI and OLA, reduced the process of spatial memory deterioration tested in the Morris water maze both in terms of the number of escape latencies and crossed quadrants.

Abnormalities in behaviour, histology and prefrontal cortical gene expression profiles relevant to schizophrenia in embryonic day 17 MAM-Exposed C57BL/6 mice.

Gestational and perinatal disruption of neural development increases the risk of developing schizophrenia (SCZ) later in life. Embryonic day 17 (E17) methylazoxymethanol (MAM) treatment leads to histological, physiological and behavioural abnormalities in post-puberty rats that model the neuropathological and cognitive deficits reported in SCZ patients. However, the validity of E17 MAM-exposed mice to model SCZ has not been explored. Here we treated E17 C57BL/6 mouse dams with various dosages of MAM. We found that this mouse strain was more vulnerable to MAM treatment than rats and there were gender differences in behavioural abnormalities, histological changes and prefrontal cortical gene expression profiles in MAM (7.5 mg/kg)-exposed mice. Both male and female MAM-exposed mice had deficits in prepulse inhibition. Female MAM-exposed mice exhibited mildly increased spontaneous locomotion activity and social recognition deficits, while male mice were normal. Consistently, only female MAM-exposed mice exhibited reduced brain weight, decreased size of prefrontal cortex (PFC) and enlarged lateral ventricles. Transcriptome analysis of the PFC revealed that there were more differentially expressed genes in female MAM-exposed mice than those in male mice. Moreover, expression of Pvalb, Arc and genes in their association networks were downregulated in the PFC of female MAM-exposed mice. These results indicate that E17 MAM-exposure in C57BL/6 mice leads to behavioural changes that model certain deficits reported in SCZ patients. MAM-exposed female mice may be used to study gene expression changes, inhibitory neural circuit dysfunction and glutamatergic synaptic plasticity deficits with a possible relation to those in the brains of SCZ patients.

Enrichment and training improve cognition in rats with cortical malformations.

Children with malformations of cortical development (MCD) frequently have associated cognitive impairments which reduce quality of life. We hypothesized that cognitive deficits associated with MCD can be improved with environmental manipulation or additional training. The E17 methylazoxymethanol acetate (MAM) exposure model bears many anatomical hallmarks seen in human MCDs as well as similar behavioral and cognitive deficits. We divided control and MAM exposed Sprague-Dawley rats into enriched and non-enriched groups and tested performance in the Morris water maze. Another group similarly divided underwent sociability testing and also underwent Magnetic Resonance Imaging (MRI) scans pre and post enrichment. A third group of control and MAM rats without enrichment were trained until they reached criterion on the place avoidance task. MAM rats had impaired performance on spatial tasks and enrichment improved performance of both control and MAM animals. Although MAM rats did not have a deficit in sociability they showed similar improvement with enrichment as controls. MRI revealed a whole brain volume decrease with MAM exposure, and an increase in both MAM and control enriched volumes in comparison to non-enriched animals. In the place avoidance task, MAM rats required approximately 3 times as long to reach criterion as control animals, but with additional training were able to reach control performance. Environmental manipulation and additional training can improve cognition in a rodent MCD model. We therefore suggest that patients with MCD may benefit from appropriate alterations in educational strategies, social interaction and environment. These factors should be considered in therapeutic strategies.

Developmental origins of adult diseases and neurotoxicity: epidemiological and experimental studies.

To date, only a small number of commercial chemicals have been tested and documented as developmental neurotoxicants. Moreover, an increasing number of epidemiological, clinical and experimental studies suggest an association between toxicant or drug exposure during the perinatal period and the development of metabolic-related diseases and neurotoxicity later in life. The four speakers at this symposium presented their research results on different neurotoxic chemicals relating to the developmental origins of health and adult disease (DOHaD). Philippe Grandjean presented epidemiological data on children exposed to inorganic mercury and methylmercury, and discussed the behavioral outcome measures as they relate to age and stage of brain development. Donald A. Fox presented data that low-dose human equivalent gestational lead exposure produces late-onset obesity only in male mice that is associated with neurodegeneration. Didima de Groot presented results on prenatal exposure of rats to methylazoxymethanol and discussed the results in light of the etiology of western Pacific amyotrophic lateral sclerosis and Parkinson-dementia complex. Merle G. Paule addressed the long-term changes in learning, motivation and short-term memory in aged Rhesus monkeys following acute 24 h exposure to ketamine during early development. Overall, these presentations addressed fundamental issues in the emerging areas of lifetime neurotoxicity testing, differential vulnerable periods of exposure, nonmonotonic dose-response effects and neurotoxic risk assessment. The results indicate that developmental neurotoxicity results in permanent changes, thus emphasizing the need to prevent such toxicity.

The genesis of epileptogenic cerebral heterotopia: clues from experimental models.

The pre-natal administration of methylazoxymethanol acetate (MAM) in rats is able to induce cerebral heterotopia that share striking similarities with those observed in human periventricular nodular heterotopia, a cerebral dysgenesis frequently associated with drug-resistant focal seizures. In the present study, we investigated the mode of neurogenesis in cerebral heterotopia of MAM-treated rats, by analyzing post-natal cytoarchitectural features and time of neurogenesis using bromodeoxyuridine immunocytochemistry. The cytoarchitectural analysis demonstrated the existence, in the early post-natal period, of white matter cellular bands in close anatomical relationship with the heterotopia, which most likely serve as a reservoir of young, migrating neurons for the newly forming heterotopia. The birth dating analysis demonstrated that the period of generation of neurons within the heterotopia and adjacent white matter bands, was extended in comparison to corticogenesis in normal rat brains. In addition, it demonstrated that the heterotopia were formed through a rather precise outside-in (for cortical and periventricular heterotopia) and dorso-ventral (for intra-hippocampal heterotopia) neurogenetic pattern. We hypothesize that the MAM-induced ablation of an early wave of cortical neurons is sufficient to alter per se the migration and differentiation of subsequently generated neurons, which in turn set the base for the formation of the different types of heterotopia. On this basis, we suggest a neurogenetic scheme for MAM-induced heterotopia that can also explain the origin and intrinsic epileptogenicity of periventricular nodular heterotopia in humans.

Cortical fiber distribution in the somatosensory cortex of rats following prenatal exposure to X-irradiation.

Exposure of fetal rats to X-irradiation at a dose of 1.5 Gy on gestational day 15 results in severe microcephaly. In these X-irradiated rats with microcephaly, a possible alteration of local circuits in the somatosensory cortex was examined using a fluorescent dye, DiI. The dye was placed at the superficial part of the transversely sectioned somatosensory cortex just beneath the pial surface of both the control and X-irradiated rats on postnatal day 11. In the controls, DiI-labeled fibers showed a layer-dependent distribution in the somatosensory cortex, whereas in the X-irradiated rats, this layer-dependent distribution of DiI-labeled fibers was distorted. Furthermore, the fasciculus observed beneath cortical layer VI (white matter), which was made of DiI-labeled axons originating from the cortical neurons, was well developed in the controls. On the other hand, in the X-irradiated rats, the fasciculus made of DiI-labeled fibers was split, reduced in number, and irregularly distributed. These observation suggest that prenatal exposure of fetal rats to X-irradiation may affect the development of local circuits in the cerebral cortex, and may also damage the axonal projection of cortical neurons to the thalamus.

Early neurobehavioral disorders in the micrencephalic offspring induced by prenatal treatment with N-methyl-N-nitrosourea or methylazoxymethanol in the rat.

Micrencephalic neonatal pups were obtained from pregnant Crj:CD (SD) rats once treated with 5 mg/kg of N-methyl-N-nitrosourea (MNU) or 40 mg/kg of methylazoxymethanol (MAM) on day 12, 13, 14 or 15 of gestation (vaginal plug = day 0). They were reared by their own mothers and were subjected to various neurobehavioral tests during the suckling period, days 0 to 22 after birth. The brain weights in the MNU- and MAM-treated pups on postnatal day 22 were significantly less than those in the control pups. These micrencephalic pups were retarded in neurobehavioral ontogeny. By several tests, each of them showed an impaired performance such as paired limb movement, clumsy locomotion or hyperreflexive reaction. These behavioral disorders appeared different according to the day of treatment, without any substantial difference between the test compounds, MNU and MAM. The findings suggest that the different neurobehavioral characteristics in the micrencephalic pups may reflect their different brain disorders induced by the test compounds given on the different period of the treatment.

Behavioral deficits in rats with minimal cortical hypoplasia induced by methylazoxymethanol acetate.

Methylazoxymethanol, a short-acting antimitotic agent, produces marked cortical hypoplasia in fetuses when injected into pregnant rats. These offspring also have increased cortical concentrations of biogenic amines associated with hyperactivity and learning deficits. In this experiment, rats with a relatively mild degree of methylazoxymethanol-induced cortical hypoplasia were studied to determine whether these neurochemical and behavioral abnormalities persisted. Sprague-Dawley pregnant rats were injected intraperitoneally on day 15 of gestation with methylazoxymethanol acetate (25 mg/kg). Total brain weight was reduced by 12% and cortical slab weight by 28% in methylazoxymethanol-exposed offspring. They were more active than control rats and showed a trend toward slower learning in a swim maze. Affected offspring had increased cortical concentrations of norepinephrine, 5-hydroxyindoleacetic acid, and glycine. There was no significant difference in the concentrations of serotonin gamma-aminobutyric acid, aspartic acid, glutamic acid, or glutamine. Methylazoxymethanol-lesioned animals with mild cortical hypoplasia remained measurably hyperactive and may serve as a model for the study of neurotransmitter and neuropathologic abnormalities associated with hyperactivity in children with microcephaly.

Effects of cytotoxic deletions of somatic sensory cortex in fetal rats.

Pregnant rats were injected on the 14th day of gestation with the cytotoxic drug methylazoxymethanol acetate. This compound causes the death of neural precursor cells that were synthesizing DNA at the time of injection. After birth, the progeny of treated mothers grew to maturity with a neocortex that was greatly reduced in area by the death of all cells, particularly at the frontal and occipital poles but at medial and lateral margins of neocortex as well. In the remaining cortex layers II through IV failed to develop. The experiment deprived growing thalamocortical axons, which innervate the somatic sensory cortex late in development, of part of their normal target area and of a substantial number of their definitive target cells. It also deprived them of any cues they might have received from these target cells migrating through them as the axons accumulate beneath the cortical plate. Anatomical experiments indicated that, despite these defects, thalamocortical axons could still colonize the sensorimotor areas and form synapses in their typically bilaminar pattern, though the outer, denser lamina of terminations occurred abnormally at the level of the apices of layer V pyramidal cell bodies. Receptive field mapping of single and multiunit responses in the somatic sensory region showed brisk responses and receptive fields of normal size. It also indicated the formation of a body map that was topographically intact except for deletions at its periphery; that is, a total map was not compressed into a smaller area. This suggests that somatic sensory thalamocortical fibers recognize only remaining cortical target cells in appropriate fields. Moreover, successful ones among them seem to recognize neighborhood relations and conserve synaptic space at the expense of those that would have innervated the deleted peripheral parts of the area. Pyramidal neurons in the remaining cortical layers and in ectopic islands of cells that had incompletely migrated from the neuroepithelium, probably on account of destruction of radial glial cell precursors, were shown by retrograde labeling to send their axons only to appropriate subcortical targets. Pyramidal neurons, though recognized as such, also adopted a variety of abnormal orientations, such as inversion, apparently in an attempt to grow apical dendrites toward major zones of synaptic terminations.

[Emulsifiers which reduce the latency of the development of colonic cancer--sodium lauryl sulfate and methylazoxymethanol].

The effect of emulsifier (nonionic surfactant) on the production of adenocarcinoma by methylazoxymethanol acetate in the large intestine of rats was studied. Following emulsifier, sodium lauryl sulfate administration, many cases of undifferentiated adenocarcinoma consisting of anaplastic glandular cells were induced in the experimental groups. Lymphatic invasion by cancer cells was found in 3 cases and metastasized to other organs in 6 cases. On the contrary, the control group (administered methylazoxymethanol acetate only) revealed well-differentiated adenocarcinoma in many cases. This fact may be due to an emulsifier used as a vehicle for the chemical, and the emulsifier might activate the character of promotion to carcinogenisity as a secondary agent. By virtue of the strong penetrating property of the emulsifier, colonial carcinogenesis seems to be enhanced.

Genetic damage caused by methylazoxymethanol acetate, the aglycone of cycasin. Analysis of sister-chromatid exchange frequency under in vivo conditions and in cell cultures.

To extend the information on the mutagenic effect of methylazoxymethanol (MAM) acetate, which has been established as a potent carcinogen, the frequency of sister-chromatid exchange (SCE) was determined in bone marrow cells of Sprague Dawley rats and in cultured rat lymphocytes. To get differentially stained chromatids under in vivo conditions, the rats received 5-bromodeoxyuridine adsorbed on activated charcoal intraperitoneally. Chromosomes were stained with fluorescence plus Giemsa. In this study a significant increase of SCE frequency after the application of various concentrations of MAM acetate could be observed both in vivo and in vivo.

A case of adult microcephaly.

A microcephalic woman who walked, spoke a few words, and was capable of some self-care, died at age 20. The brain weighed 260 g--the equivalent of that of an infant of eight months' gestation. The remarkable neuropathological features in our case included very small cerebral hemispheres, normal cerebral cortex, myelinated white matter, and large neuronal heterotopias situated along the ventricular walls and deep in the white matter adjacent to the intact nuclei of the basal ganglia, amygdala, and thalamus. The brainstem and cerebellum were relatively less reduced in size and showed normal structure and no heterotopias. In rats, microcephaly can be induced experimentally by giving carcinogens during sensitive periods of embryogenesis. Here, the microcephaly appears to result from necrosis of the neuroblasts lining the ventricles, and from a concomitant reduction in DNA synthesis. The pathogenesis of the microcephaly in our patient and others remains undefined. Our study shows that a great discrepancy may exist between brain size and level of function.