Marialaura Santarelli

Spatial analysis reveals alterations of parvalbumin- and calbindin-positive local circuit neurons in the cerebral cortex of mutant mdx mice

The aim of the present study was to investigate the spatial organization of selected populations of local circuit neurons in the cerebral cortex of the mutant mdx mouse, an acknowledged model of Duchenne Muscular Dystrophy. To this purpose, we quantified and compared the distribution of parvalbumin- and calbindin-positive neurons in the motor, somatosensory, visual, and anterior cingulate cortices of wild-type and mdx mice. The methodological approach was based on generation of two-dimensional Voronoi polygons from digital charts of the cell populations visualized immunohistochemically. Polygon areas were then analyzed and the derived coefficients of variation were statistically compared. Using this strategy, we were able to reveal, in mdx mice, changes involving both the above populations of interneurons. These changes were evident in the motor and anterior cingulate cortices but not in the somatosensory and visual cortices. In addition, the changes of coefficients of variation were area-specific in the cortex of mdx mice. The values increased in the motor cortex and decreased in the anterior cingulate cortex with respect to the corresponding values of wild-type animals. The present findings point out widespread alterations in the mdx cortex involving also areas not primarily related to sensorimotor integration. In addition, we demonstrate that cortical alterations of the local circuit machinery are characterized in mdx mice by individual regional differences.

Multifaceted alterations of the thalamo-cortico-thalamic loop in adult rats prenatally exposed to ethanol

The thalamo-cortico-thalamic loop was investigated in adult rats exposed to ethanol during the last week of fetal life. Animals underwent either cortical or thalamic injections of lectin-conjugated horseradish peroxidase. Results demonstrate that prenatal exposure to ethanol causes permanent changes in the thalamocortical circuits. Alterations of thalamo-cortical and cortico-thalamic projections are concentrated at the level of axon terminal fields. The most severe thalamic damage is observed in the anterior intralaminar and midline nuclei; crossed cortico-thalamic projections also appear to be severely impaired. In the cortex, the damage to thalamic terminals displays a medio-lateral gradient of increasing severity through sensori-motor areas, with the lateral fields more impaired. Cells of origin of thalamo-cortical and cortico-thalamic projections are less affected by prenatal ethanol exposure: in the thalamus and layer 5 of sensori-motor cortex labeled cells exhibit normal values of areal numeric density. Conversely, cortico-thalamic neurons of layer 6, especially in the lateral agranular sensori-motor field, display smaller values of areal density than those of normal animals. Possible mechanisms underlying the establishment of these abnormalities are discussed.

Cortical and brainstem neurons containing calcium‐binding proteins in a murine model of Duchenne's muscular dystrophy: Selective changes in the sensorimotor cortex

In the muscular dystrophic (mdx) mouse, which is characterized by deficient dystrophin expression and provides a model of Duchennes muscular dystrophy, we previously demonstrated marked central nervous system alterations and in particular a quantitative reduction of corticospinal and rubrospinal neurons and pathologic changes of these cells. Prompted by these findings and in view of the relations between calcium ions and dystrophin, we analyzed with immunohistochemistry the neurons containing the calcium‐binding proteins parvalbumin, calbindin D28k, and calretinin in cortical areas and brainstem nuclei of mdx mice. In the sensorimotor cortex, parvalbumin‐positive and calbindin‐positive neurons, which represent a subset of cortical interneurons, were significantly more numerous in mdx mice than in wild‐type ones. In addition, the laminar distribution of parvalbumin‐positive neurons in the motor and somatosensory cortical areas of mdx mice was altered with respect to wild‐type animals. No alterations in the number and distribution were found in the parvalbumin‐ or calbindin‐expressing cell populations of the visual and anterior cingulate cortices of mdx mice. The pattern of calretinin immunoreactivity was normal in all investigated cortical areas. The cell populations containing either calcium‐binding protein were similar in brainstem nuclei of mdx and wild‐type mice. The present findings demonstrated selective changes of subsets of interneurons in the motor and somatosensory cortical areas of mdx mice. Therefore, the data showed that, in the cortices of these mutant animals, the previously demonstrated pathologic changes of corticospinal cell populations are accompanied by marked alterations in the local circuitry. J. Comp. Neurol. 456:48–59, 2003.

The organisation of spinal projecting brainstem neurons in an animal model of muscular dystrophy: A retrograde tracing study on mdx mutant mice

Previous studies we performed on the mdx mouse demonstrated marked central nervous system alterations in this model of human Duchenne muscular dystrophy, such as reduction in number and pathological changes of cortico-spinal neurons. Prompted by these findings we extended the survey of the mdx brain to the major brainstem-descending pathways: the rubro-, vestibulo-, reticulo-, and raphe-spinal projections. Horseradish peroxidase microinjections were performed in the cervical spinal cord of mdx and control mice. The rubro-spinal neurons were found to be significantly reduced in mutants compared to controls. The vestibulo-spinal, reticulo-spinal, and raphe-spinal cell populations, though less numerous in mdx than in control mice, were instead substantially spared. Our data further unveil the selective nature of mdx brain damage indicating a marked and selective involvement of the highest centers for motor control.

Crossed thalamo-cortical and cortico-thalamic projections in adult mice

The crossed thalamo-cortical and cortico-thalamic connections of the mouse are drawn using the tracer wheat germ agglutinin-horseradish peroxidase. After injections in the frontal cortex of the right hemisphere cells labeled retrogradely and axons labeled anterogradely are observed in the thalamus ipsilateral and contralateral to the cortical injections. The retrograde and anterograde labeling in the contralateral thalamus is less intense than ipsilaterally and involves the mediodorsal, ventral medial, central medial, and paracentral nuclei. Crossed fronto-thalamic axons reach more lateral regions than those containing contralateral thalamo-frontal neurons. Our results demonstrate that the thalamo-cortical system of mice has a bilateral component. The functional significance of this pathway and analogies with crossed thalamo-cortical connections in other species are discussed.

Modifications of thalamo-cortical circuitry in rats prenatally exposed to ethanol.

THE present study aimed to investigate the organization of thalamo-cortical connections in adult rats exposed to ethanol during the last week of foetal life. Animals underwent thalamic injections of lectin-conjugated HRP. Results demonstrate that the thalamic-recipient zone of sensorimotor cortex is significantly thinner in ethanol- exposed than in control cases. Animals exposed to ethanol also display aberrant thalamo-cortical terminations in layer 5a. Neurones of origin of cortico-thalamic projections are normally located in layers 5 and 6; they appear quantitatively comparable in control and ethanolexposed cases. Developmental alterations underlying the establishment of anomalous thalamo-cortical relationships are discussed.

Bihemispheric organization of amygdalo-cortical projections in the rat

Two fluorescent dyes were injected symmetrically into the prefrontal cortices of the left and right hemisphere. A consistent number of amygdaloid neurons was labeled from the injection in the contralateral cortex. Each nucleus of the basolateral amygdaloid complex showed a specific pattern of contralaterality. The highest degree of bihemisphericity was observed in the ventral basolateral nucleus. A significant number of neurons connected with the cortex of both hemispheres by means of axon collaterals was observed in the anterior basolateral nucleus. These anatomical findings indicate that the basolateral amygdaloid complex could act as subcortical link for interhemispheric communication.

Alterations of the thalamo-cortical system in rats prenatally exposed to ethanol are prevented by concurrent administration of acetyl- l-carnitine

We previously demonstrated that adult rats prenatally exposed to ethanol display permanent damages of thalamo-cortical connections [18,19,33]. Here the effect of simultaneous administration of ethanol and acetyl-L-carnitine has been investigated. Adult animals underwent cortical or thalamic injections of horseradish peroxidase and both anterograde and retrograde thalamic and cortical labeling have been analyzed. Ethanol-induced changes of thalamo-cortical circuits are prevented by concurrent administration of acetyl-L-carnitine. Possible mechanisms underlying this effect are discussed.

An optimised procedure for prenatal ethanol exposure with determination of its effects on central nervous system connections.

We describe the protocol set-up to investigate an experimental model of foetal alcohol syndrome in the rat. The protocol has been devised to expose specific cell populations of the central nervous system to ethanol during their neurogenesis and has been applied to the study of diencephalo-telencephalic connections. We were able to demonstrate specific permanent changes of the adult thalamo-cortical circuitry. Our protocol can be applied to study other aspects of central nervous system-ethanol interactions, such as neurotransmitter and receptor patterns. It can also represent a useful tool to test the effects of different diets to prevent nutritional deficiencies and the efficacy of drug treatments to prevent foetal alcohol syndrome. We have shown in fact that ethanol-induced thalamo-cortical alterations are partially prevented by concurrent administration of acetyl-L-carnitine. Finally, the present protocol can be used to investigate the effects of ethanol exposure on the development of different brain structures. To this purpose, the gestational period for ethanol exposure must be chosen according to the peak of neurogenesis for the investigated structure.

A simple pressure microinjecting system for delivery of small substance volumes to the brain: application to the developmental study of thalamo-cortical projections in foetal and neonatal rats

We describe a reliable and inexpensive method for placing injections of anatomical tracers into the brain of lower mammals. The pressure microinjecting system we developed is specifically designed to deliver very small amount of substances. The injecting portion of the system is relatively easy to assemble and can be repeatedly used for multiple experimental sessions. The system has been validated with experiments of multiple fluorescent retrograde tracing. In these experiments the populations of thalamo-cortical neurons were consistently labeled by the tracers injected bilaterally and symmetrically in the cortex of foetal and neonatal rats.