Prenatal methylazoxymethanol treatment in rats produces brain abnormalities with morphological similarities to human developmental brain dysgeneses.

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.

Dysplastic neocortex and subcortical heterotopias in methylazoxymethanol-treated rats: an intracellular study of identified pyramidal neurones.

Intracellular recordings were obtained using biocytin-filled electrodes from 78 neurones located in both dysplastic neocortex and subcortical heterotopic aggregates in a model of neuronal migration disorder induced in rats by means of a double methylazoxymethanol injection given on embryonic day 15. Both regular spiking and intrinsically bursting pyramidal neurones were found in all of the examined structures and were synaptically activated by subcortical stimulation. In a neuronal subpopulation (22%) located in the neocortex as well as in the subcortical heterotopic aggregates, the injection of depolarising current pulses elicited aberrant firing patterns, consisting of repetitive bursts of APs that gradually increased in duration and eventually merged in a long-lasting discharge. The gradual development of this 'excessive' bursting behaviour suggests a progressive run-down of the slow components of the hyperpolarising afterpotential.

Altered connections between neocortical and heterotopic areas in methylazoxymethanol-treated rat.

We are currently investigating various treatments which could determine, in the rat brain, structural abnormalities mimicking those reported in human brain dysgeneses. We can induce the formation of neuronal heterotopia in the progeny of rats by means of a double injection of the cytotoxic agent methylazoxymethanol acetate (MAM) on embryonic day 15. We have now investigated the anatomical connections of these heterotopia by means of anterograde and retrograde tract tracing techniques. The induced heterotopia along the border of the lateral ventricles shared common anatomical features with the periventricular nodules in human periventricular or subcortical nodular heterotopia (PNH). The tract tracing data demonstrated the existence of reciprocal connections between the neuronal heterotopia and the ipsilateral and contralateral cortical areas, and the presence of abnormal cortico-hippocampal and cortico-cortical connections. On the basis of the connectivity patterns, it may be speculated that some cells in the heterotopia could be neurons originally committed to the cortex, that were interrupted in their migration by the MAM treatment. Given the common morphological features seen in human PNH and MAM-induced brain heterotopia, the anatomical and developmental analysis of MAM-treated rats may shed light on the mechanisms by which human brain dysgeneses develop in human patients.

A reticuloreticular commissural pathway in the rat thalamus.

To further characterize the communication between the thalami of the two hemispheres, a connection linking the rostral reticular nuclei of the two thalamic sides was investigated in the rat by retrograde and anterograde tracing. The rostral reticular nucleus can be divided into a medial region, with densely packed fusiform neurons, and a lateral region, with less densely packed, polymorphic neurons. After injections of Fluorogold (FG) in the medial region, retrogradely labeled, small fusiform neurons were found in the corresponding contralateral region. The retrograde labeling data were confirmed by the anterograde-tracing experiments. Thin, beaded axons, anterogradely labeled after injection of biocytin or biotinylated dextranamine in the medial region, innervate the corresponding region in the contralateral reticular nucleus. The present data suggest the existence of a commissural pathway specifically devoted to the crosstalk between the rostral reticular nuclei of the two thalamic sides. The commissural gamma aminobutyric acid (GABA)-ergic input on the GABAergic neurons of the rostral reticular nucleus could modulate the generation of sleep spindles. The reticuloreticular pathway may, moreover, synchronize the diffuse modulatory effect of the rostral reticular nucleus on nonprimary cortical areas through the bilateral projections of the nucleus to the ventromedial, intralaminar, and anterior thalamic nuclei.

The relationship of calbindin-containing neurons with substance P, Leu-enkephalin and cholecystokinin fibres: an immunohistochemical study in the rat thalamus.

In the rat thalamus, immunoreactivity for the calcium binding protein calbindin (Cb) is mostly confined to neuronal cell bodies, sometimes revealing proximal dendrites, of the midline, intralaminar and posterior regions. Substance P (SP)-, cholecystokinin (CCK)- and Leu-enkephalin (L-ENK)-immunoreactive (ir) elements in the thalamus are fibre-like structures, intermingled with punctate elements probably representing axonal arborizations and their synaptic boutons. These peptidergic fibres are unevenly distributed in several thalamic domains, including the areas that contain Cb-ir neurons. The relationship between Cb-ir cell bodies and these three different peptidergic systems of thalamic innervation was studied with immunohistochemistry. Single-labelling experiments on adjacent sections and double immunostaining on the same section were performed. A considerable overlap between Cb-ir perikarya and SP-ir fibres was found in most thalamic nuclei. In particular, in the intralaminar nuclei and posterior complex, SP-ir punctate elements were frequently observed in close proximity to Cb-ir cell bodies and dendrites. On the other hand, no consistent topographical correspondence between Cb-ir perikarya and CCK- or L-ENK-ir fibres was evident. Altogether, the present data suggest a selective anatomical and, possibly, functional relationship between SP and Cb in at least a subpopulation of rat thalamic neurons.