Giovanna Ponti

Genesis of Neuronal and Glial Progenitors in the Cerebellar Cortex of Peripuberal and Adult Rabbits

Adult neurogenesis in mammals is restricted to some brain regions, in contrast with other vertebrates in which the genesis of new neurons is more widespread in different areas of the nervous system. In the mammalian cerebellum, neurogenesis is thought to be limited to the early postnatal period, coinciding with end of the granule cell genesis and disappearance of the external granule cell layer (EGL). We recently showed that in the rabbit cerebellum the EGL is replaced by a proliferative layer called ‘subpial layer’ (SPL) which persists beyond puberty on the cerebellar surface. Here we investigated what happens in the cerebellar cortex of peripuberal rabbits by using endogenous and exogenously-administered cell proliferation antigens in association with a cohort of typical markers for neurogenesis. We show that cortical cell progenitors extensively continue to be generated herein. Surprisingly, this neurogenic process continues to a lesser extent in the adult, even in the absence of a proliferative SPL. We describe two populations of newly generated cells, involving neuronal cells and multipolar, glia-like cells. The genesis of neuronal precursors is restricted to the molecular layer, giving rise to cells immunoreactive for GABA, and for the transcription factor Pax2, a marker for GABAergic cerebellar interneuronal precursors of neuroepithelial origin that ascend through the white matter during early postnatal development. The multipolar cells are Map5+, contain Olig2 and Sox2 transcription factors, and are detectable in all cerebellar layers. Some dividing Sox2+ cells are Bergmann glia cells. All the cortical newly generated cells are independent from the SPL and from granule cell genesis, the latter ending before puberty. This study reveals that adult cerebellar neurogenesis can exist in some mammals. Since rabbits have a longer lifespan than rodents, the protracted neurogenesis within its cerebellar parenchyma could be a suitable model for studying adult nervous tissue permissiveness in mammals.

Cellular composition and cytoarchitecture of the rabbit subventricular zone and its extensions in the forebrain

Persistent neurogenic sites, harboring neurogenic progenitor cells, which give rise to neuronal precursors throughout life, occur in different mammals, including humans. The telencephalic subventricular zone (SVZ) is the most active adult neurogenic site. Despite remarkable knowledge of its anatomical and cellular composition in rodents, detailed arrangement of SVZ in other mammals is poorly understood, yet comparative studies suggest that differences might exist. Here, by analyzing the cellular composition/arrangement in the SVZ of postnatal, young, and adult rabbits, we found a remarkably heterogeneous distribution of its chain and glia compartments. Starting from postnatal stages, this heterogeneity leads to a distinction between a ventricular SVZ and an abventricular SVZ, whereby the former contains small chains and isolated neuroblasts and the latter is characterized by large chains and a loose astrocytic meshwork. In addition to analysis of the SVZ proper, attention has been focused on its extensions, called parenchymal chains. Anterior parenchymal chains are compact chains surrounded by axon bundles and frequently establish direct contact with blood vessels. Posterior parenchymal chains are less compact, being squeezed between gray and white matter. In the shift from neonatal to adult rabbit SVZ, chains occur very early, both in the SVZ and within the brain parenchyma. Comparison of these results with the pattern in rodents reveals different types of chains, displaying a variety of relationships with glia or other substrates in vivo, an issue that might be important in understanding differences in the adaptation of persistent germinative layers to different mammalian brain anatomies. J. Comp. Neurol. 498:491–507, 2006.

Comparative expression profiles of ShcB and ShcC phosphotyrosine adapter molecules in the adult brain

Shc family of adaptor molecules has been demonstrated to play an important role during the transition from proliferating neural stem cells to postmitotic neurons. Previous studies from our group demonstrated a progressive decrease of ShcA levels occurring in coincidence with the end of embryonic neurogenesis and neuronal maturation, being ShcB and ShcC the major Shc molecules expressed in the mature brain. A growing body of evidence indicates that ShcB and ShcC are neuronal specific molecules exerting important roles in neuronal survival and phenotypic stability thus becoming potential attracting target molecules for development of drugs for interfering with brain demises. Here, we examine the expression pattern of ShcB and ShcC in neuronal populations composing the adult central and peripheral nervous system, in order to better elucidate their roles in vivo. We found a heterogeneous and peculiar presence and subcellular localization of ShcB and ShcC in specific neuronal populations, enlightening a potential specific requirement of these two molecules in the survival/maintenance of defined neuronal subtypes.

Lineage progression from stem cells to new neurons in the adult brain ventricular-subventricular zone.

It has been estimated that ~10,000 new neurons are generated daily in the mouse ventricular-subventricular zone (V-SVZ), an extensive germinal niche present in the walls of the lateral brain ventricles in many adult mammals. Neural stem cells, a subpopulation of GFAP-expressing astrocytes (B1 cells), contact the ventricle with a small apical process surrounded by ependymal cells forming pinwheel-like structures. B1 cells generate intermediate progenitors (C cells), which differentiate into neuroblasts (A cells). A cells also divide and migrate out of the V-SVZ along the rostral migratory stream into the olfactory bulb, where they differentiate into local interneurons. A full understanding of this process requires precise information of the dynamics of proliferation of V-SVZ progenitor populations and how many times each divides. This information is now available for the adult mouse brain. The reporter protein in hGFAP::GFP mice was used to identify B1 cells and immunolabeling for Ascll, and DCX was used to identify C and A cells, respectively. Of the Ascll + cells, only 3.1+/−0.6% were also GFP+, while 15.9+/−1.6% were DCX+, indicating a small overlap between populations, possibly associated to transitional stages between cell types. We used whole-mount preparations, which provide an en face view of the lateral ventricular surface, to analyze the entire neurogenic niche, minimizing the effect of rostral migration of proliferating progenitors on our quantifications. Using a combination of thymidine analogs (CldU and EdU) and Lineage progression from stem cells to new neurons in the adult brain ventricular-subventricular zone

Neural-specific inactivation of ShcA functions results in anatomical disorganization of subventricular zone neural stem cell niche in the adult brain.

Shc(s) family of adaptor molecules has been implicated in several physiological functions. In particular, our previous studies have shown major roles in the mechanisms that control the transition from proliferating neural stem cells (NSCs) to postmitotic neurons in the mammalian brain. In the adult brain, ShcA expression is mainly restricted to a subpopulation of cells in the subventricular zone (SVZ) neurogenic area, enlightening a potential role for this molecule in the establishment/maintenance of this adult NSC niche. In order to investigate this matter, here we took advantage of Cre/lox technology with the purpose of interfering with (or delete) ShcA function in nestin-expressing neural progenitors in vivo. Our analyses revealed signs of anatomical disorganization in the adult brain at the boundary between the striatum and the corpus callosum and reduced thickness both at the ventricular level and through the rostral migratory stream. Analysis of cell proliferation and cell death unveiled a prominent reduction of the former and no substantial alterations of the latter. Ultrastructural studies showed SVZ anatomical disarray and manifest variation in the SVZ cell type composition. In conclusion, these results provide evidence for a role of ShcA in the assembly and/or maintenance of the SVZ NSC niche in the adult brain.

Kisspeptin innervation of the hypothalamic paraventricular nucleus: sexual dimorphism and effect of estrous cycle in female mice

The hypothalamic paraventricular nucleus (PVN) is the major autonomic output area of the hypothalamus and a critical regulatory center for energy homeostasis. The organisms energetic balance is very important for both the regular onset of puberty and regulation of fertility. Several studies have suggested a relationship among neural circuits controlling food intake, energy homeostasis and the kisspeptin peptide. The kisspeptin system is clustered in two main groups of cell bodies [the anterior ventral periventricular region (AVPV) and the arcuate nucleus (ARC)] projecting mainly to gonadotropin‐releasing hormone (GnRH) neurons and to a few other locations, including the PVN. In the present study, we investigated the distribution of the kisspeptin fibers within the PVN of adult CD1 mice. We observed a significant sexual dimorphism for AVPV and ARC, as well as for the PVN innervation. Kisspeptin fibers showed a different density within the PVN, being denser in the medial part than in the lateral one; moreover, in female, the density changed, according to different phases of the estrous cycle (the highest density being in estrus phase). The presence of a profound effect of estrous cycle on the kisspeptin immunoreactivity in AVPV (with a higher signal in estrus) and ARC, and the strong co‐localization between kisspeptin and NkB only in ARC and not in PVN suggested that the majority of the kisspeptin fibers found in the PVN might arise directly from AVPV.

Immuno-electromicroscopic approach for the study of neural stem cell niches

Immuno-electromicroscopic approach for the study of neural stem cell niches L. Bonfanti & P. Aimar & G. Ponti & N. Canalia Published online: 7 August 2008 # Springer Science + Business Media B.V. 2008

Sex Steroids and Adult Neurogenesis in the Ventricular-Subventricular Zone

The forebrain ventricular-subventricular zone (V-SVZ) continuously generates new neurons throughout life. Neural stem cells (type B1 cells) along the lateral ventricle become activated, self-renew, and give rise to proliferating precursors which progress along the neurogenic lineage from intermediate progenitors (type C cells) to neuroblasts (type A cells). Neuroblasts proliferate and migrate into the olfactory bulb and differentiate into different interneuronal types. Multiple factors regulate each step of this process. Newly generated olfactory bulb interneurons are an important relay station in the olfactory circuits, controlling social recognition, reproductive behavior, and parental care. Those behaviors are strongly sexually dimorphic and changes throughout life from puberty through aging and in the reproductive age during estrous cycle and gestation. Despite the key role of sex hormones in regulating those behaviors, their contribution in modulating adult neurogenesis in V-SVZ is underestimated. Here, we compare the literature highlighting the sexual dimorphism and the differences across the physiological phases of the animal for the different cell types and steps through the neurogenic lineage.

Early postnatal genistein administration permanently affects nitrergic and vasopressinergic systems in a sex-specific way

Genistein (GEN) is a natural xenoestrogen (isoflavonoid) that may interfere with the development of estrogen-sensitive neural circuits. Due to the large and increasing use of soy-based formulas for babies (characterized by a high content of GEN), there are some concerns that this could result in an impairment of some estrogen-sensitive neural circuits and behaviors. In a previous study, we demonstrated that its oral administration to female mice during late pregnancy and early lactation induced a significant decrease of nitric oxide synthase-positive cells in the amygdala of their male offspring. In the present study, we have used a different experimental protocol mimicking, in mice, the direct precocious exposure to GEN. Mice pups of both sexes were fed either with oil, estradiol or GEN from birth to postnatal day 8. Nitric oxide synthase and vasopressin neural systems were analyzed in adult mice. Interestingly, we observed that GEN effect was time specific (when compared to our previous study), sex specific, and not always comparable to the effects of estradiol. This last observation suggests that GEN may act through different intracellular pathways. Present results indicate that the effect of natural xenoestrogens on the development of the brain may be highly variable: a plethora of neuronal circuits may be affected depending on sex, time of exposure, intracellular pathway involved, and target cells. This raises concern on the possible long-term effects of the use of soy-based formulas for babies, which may be currently underestimated.

The Rabbit Subventricular Zone (SVZ): An Ultrastructural and Immunocytochemical Study

The subventricular zone (SVZ) is a germinative layer persisting throughout life within the mammalian telencephalon (Gage, 2000). In all species studied, the SVZ generates neuronal precursors which migrate toward the olfactory bulb, where they differentiate into interneurons (Lois and Alvarez-Buylla, 1994). In laboratory rodents, cell migration occurs in the form of chains of neuroblasts wrapped with an astrocytic sheath referred to as ‘glial tubes’ (Bonfanti and Theodosis, 1994; Peretto et al., 1997). Studies carried out in Primates have hypothesized that some SVZ-generated cells could also reach other cortical regions (Gould et al., 1999; Bernier et al., 2002). In a recent work performed on the rabbit telencephalon (Luzzati et al., 2003) we described the existence of ‘parenchymal chains’ localized between the SVZ and the cerebral cortex, which could theoretically be linked with such hypothesis. In the present study, we addressed the issue of rabbit SVZ internal arrangement. Using confocal and electron microscopy, we performed a detailed analysis of the young and adult rabbit SVZ, to identify its cell types and to investigate their mutual relationships.