Testosterone and estradiol differentially affect cell proliferation in the subventricular zone of young adult gonadectomized male and female rats.

Steroid hormones are important players to regulate adult neurogenesis in the dentate gyrus of the hippocampus, but their involvement in the regulation of the same phenomenon in the subventricular zone (SVZ) of the lateral ventricles is not completely understood. Here, in male rats, we tested the existence of activational effects of testosterone (T) on cell proliferation in the adult SVZ. To this aim, three groups of male rats: castrated, castrated and treated with T, and controls were treated with 5-bromo-2'-deoxyuridine (BrdU) and killed after 24h. The density of BrdU-labeled cells was significantly lower in castrated animals in comparison to the other two groups, thus supporting a direct correlation between SVZ proliferation and levels of circulating T. To clarify whether this effect is purely androgen-dependent, or mediated by the T metabolites, estradiol (E2) and dihydrotestosterone (DHT), we evaluated SVZ proliferation in castrated males treated with E2, DHT and E2+DHT, in comparison to T- and vehicle-treated animals, and sham-operated controls. The stereological analysis demonstrated that E2 and T, but not DHT, increase proliferation in the SVZ of adult male rats. Quantitative evaluation of cells expressing the endogenous marker of cell proliferation phosphorylated form of Histone H3 (PHH3), or the marker of highly dividing SVZ progenitors Mash1, indicated the effect of T/E2 is mostly restricted to SVZ proliferating progenitors. The same experimental protocol was repeated on ovariectomized female rats treated with E2 or T. In this case, no statistically significant difference was found among groups. Overall, our results clearly show that the gonadal hormones T and E2 represent important mediators of cell proliferation in the adult SVZ. Moreover, we show that such an effect is restricted to males, supporting adult neurogenesis in rats is a process differentially modulated in the two sexes.

From chemical neuroanatomy to an understanding of the olfactory system.

The olfactory system is the appropriate model for studying several aspects of neuronal physiology spanning from the developmental stage to neural network remodelling in the adult brain. Both the morphological and physiological understanding of this system were strongly supported by classical histochemistry. It is emblematic the case of the Olfactory Marker Protein (OMP) staining, the first, powerful marker for fully differentiated olfactory receptor neurons and a key tool to investigate the dynamic relations between peripheral sensory epithelia and central relay regions given its presence within olfactory fibers reaching the olfactory bulb (OB). Similarly, the use of thymidine analogues was able to show neurogenesis in an adult mammalian brain far before modern virus labelling and lipophilic tracers based methods. Nowadays, a wealth of new histochemical techniques combining cell and molecular biology approaches is available, giving stance to move from the analysis of the chemically identified circuitries to functional research. The study of adult neurogenesis is indeed one of the best explanatory examples of this statement. After defining the cell types involved and the basic physiology of this phenomenon in the OB plasticity, we can now analyze the role of neurogenesis in well testable behaviours related to socio-chemical communication in rodents.

Integration and sensory experience-dependent survival of newly-generated neurons in the accessory olfactory bulb of female mice.

Newborn neurons generated by proliferative progenitors in the adult subventricular zone (SVZ) integrate into the olfactory bulb circuitry of mammals. Survival of these newly-formed cells is regulated by the olfactory input. The presence of new neurons in the accessory olfactory bulb (AOB) has already been demonstrated in some mammalian species, albeit their neurochemical profile and functional integration into AOB circuits are still to be investigated. To unravel whether the mouse AOB represents a site of adult constitutive neurogenesis and whether this process can be modulated by extrinsic factors, we have used multiple in vivo approaches. These included fate mapping of bromodeoxyuridine-labelled cells, lineage tracing of SVZ-derived enhanced green fluorescent protein-positive engrafted cells and neurogenesis quantification in the AOB, in both sexes, as well as in females alone after exposure to male-soiled bedding or its derived volatiles. Here, we show that a subpopulation of SVZ-derived neuroblasts acquires proper neurochemical profiles of mature AOB interneurons. Moreover, 3D reconstruction of long-term survived engrafted neuroblasts in the AOB confirms these cells show features of fully integrated neurons. Finally, exposure to male-soiled bedding, but not to its volatile compounds, significantly increases the number of new neurons in the AOB, but not in the main olfactory bulb of female mice. These data show SVZ-derived neuroblasts differentiate into new functionally integrated neurons in the AOB of young and adult mice. Survival of these cells seems to be regulated by an experience-specific mechanism mediated by pheromones.

BDNF/ TrkB interaction regulates migration of SVZ precursor cells via PI3-K and MAP-K signalling pathways.

Neuroblasts born in the subventricular zone (SVZ) migrate along the rostral migratory stream, reaching the olfactory bulb (OB) where they differentiate into local interneurons. Several extracellular factors have been suggested to control specific steps of this process. The brain-derived neurotrophic factor (BDNF) has been demonstrated to promote morphological differentiation and survival of OB interneurons. Here we show that BDNF and its receptor TrkB are expressed in vivo throughout the migratory pathway, implying that BDNF might also mediate migratory signals. By using in vitro models we demonstrate that BDNF promotes migration of SVZ neuroblasts, acting both as inducer and attractant through TrkB activation. We show that BDNF induces cAMP response element-binding protein (CREB) activation in migrating neuroblasts via phosphatidylinositol 3-kinase (PI3-K) and mitogen-activated protein kinase (MAP-K) signalling. Pharmacological blockade of these pathways on SVZ explants significantly reduces CREB activation and impairs neuronal migration. This study identifies a function of BDNF in the SVZ system, which involves multiple protein kinase pathways leading to neuroblast migration.

Maternal deprivation and early handling affect density of calcium binding protein-containing neurons in selected brain regions and emotional behavior in periadolescent rats.

Adverse early life experiences can induce neurochemical changes that may underlie modifications in hypothalamic-pituitary-adrenal axis responsiveness, emotionality and cognition. Here, we investigated the expression of the calcium binding proteins (CBPs) calretinin, calbindin and parvalbumin, which identify subpopulations of GABAergic neurons and serve important functional roles by buffering intracellular calcium levels, following brief (early handling) and long (maternal deprivation) periods of maternal separation, as compared with non-handled controls. CBP-expressing neurons were analyzed in brain regions related to stress and anxiety. Emotionality was assessed in parallel using the social interaction test. Analyses were carried out at periadolescence, an important phase for the development of brain areas involved in stress responses. Our results indicate that density of CBP-immunoreactive neurons decreases in the paraventricular region of deprived rats but increases in the hippocampus and lateral amygdala of both early-handled and deprived rats when compared with controls. Emotionality is reduced in both early-handled and deprived animals. In conclusion, early handling and deprivation led to neurochemical and behavioral changes linked to stress-sensitive brain regions. These data suggest that the effects of early experiences on CBP containing neurons might contribute to the functional changes of neuronal circuits involved in emotional response.

Expression of the secreted factors noggin and bone morphogenetic proteins in the subependymal layer and olfactory bulb of the adult mouse brain.

The antagonism between noggin and the bone morphogenetic proteins (BMPs) plays a key role during CNS morphogenesis and differentiation. Recent studies indicate that these secreted factors are also widely expressed in the postnatal and adult mammalian brain in areas characterized by different types of neural plasticity. In particular, significant levels of noggin and BMP expression have been described in the rodent olfactory system. In the mammalian forebrain, the olfactory bulb (OB) and associated subependymal layer (SEL) are documented as sites of adult neurogenesis. Here, using multiple approaches, including the analysis of noggin-LacZ heterozygous mice, we report the expression of noggin and two members of the BMP family, BMP4 and BMP7, in these regions of the adult mammalian forebrain. We observe that along the full extent of the SEL, from the lateral ventricle to the olfactory bulb, noggin and BMP4 and 7 are mainly associated with the astrocytic glial compartment. In the OB, BMP4 and 7 proteins remain primarily associated with the SEL while strong noggin expression was also found in cells located in different OB layers (i.e. granule, external plexiform, glomerular layers). Taken together our data lead us to hypothesize that within the SEL the antagonism between noggin and BMPs, both produced by the glial tubes, act through autocrine/paracrine inductive mechanisms to maintain a neurogenetic environment all the way from the lateral ventricle to the olfactory bulb. In the OB, their expression patterns suggest multiple regulatory roles on the unusual neural plasticity exhibited by this region.

Glia-independent chains of neuroblasts through the subcortical parenchyma of the adult rabbit brain.

In the brains of adult mammals long-distance cell migration of neuronal precursors is known to occur in the rostral migratory stream, involving chains of cells sliding into astrocytic glial tubes. By combining immunocytochemistry for polysialylated neural cell adhesion molecule (PSA-NCAM), neuronal and glial antigens, endogenous and exogenously administered cell-proliferation markers, and light and electron microscopy 3D reconstructions, we show that chains of newly generated neuroblasts exist both inside and outside the subventricular zone of adult rabbits. Two groups of chains were detectable within the mature brain parenchyma: anterior chains, into the anterior forceps of the corpus callosum, and posterior chains, close to the external capsule. Parenchymal chains were not associated with any special glial structures, thus coming widely in contact with the mature nervous tissue, including unmyelinated/myelinated fibers, astrocytes, neurons, and oligodendrocytes. These chains of cells, unlike those in the subventricular zone, do not display cell proliferation, but they contain BrdUrd administered several weeks before. Telencephalic areas, such as the putamen, amygdala, claustrum, and cortex, adjacent to the chains harbor numerous PSA-NCAM-positive cells. The counting of newly generated cells in these areas shows small differences in comparison with others, and a few cells double-labeled for BrdUrd/PSA-NCAM (after 1-month survival) and for BrdUrd/NeuN (after 2 months) were detectable. These results demonstrate the occurrence of glial-independent chains of migrating neuroblasts, which directly contact the mature brain parenchyma of adult mammals. These chains could provide a possible link between the adult germinative layers and a very low-rate/long-term process of cell addition in the telencephalon.

Sexually dimorphic neurogenesis is topographically matched with the anterior accessory olfactory bulb of the adult rat.

The accessory olfactory bulb (AOB) is a sexually dimorphic structure of the vomeronasal system, which plays a role in the control of sexual behaviors. In adult rats, we have demonstrated previously that the migrating neuroblasts of the subependymal layer (SEL) directed to the main olfactory bulb (MOB) also reach the AOB. To tackle the relation between sexual dimorphism and targeted cell migration, we quantified the neo-neurogenesis in the AOB of adult rats of both sexes. Our results confirm a morphological sexual dimorphism in the AOB granular layer volumes. We showed that the number of newly generated cells reaching the AOB in both sexes was considerable, even if lower than those directed to the MOB. Moreover, we demonstrated that the rate of neurogenesis in the anterior AOB of the two sexes was significantly different.

Aminoacyl-histidine dipeptides in the glial cells of the adult rabbit forebrain.

The mammalian nervous system contains high amounts of the aminoacyl-histidine dipeptides carnosine and homocarnosine. In the brain, they prevalently occur mainly in glial and ependymal cells, their role(s) still remaining obscure. In vitro studies indicate that these molecules exert diverse protective effects, and in vivo they are frequently associated with extracellular fluid compartments. Recently, carnosine-like immunoreactivity has been found in the subependymal layer (SEL) of adult rodents, a region endowed with persistent cell proliferation and migration. Unlike rodents, the SEL of the rabbit has a persistent olfactory ventricle. We show here that the morphologic organization of the SEL is different in these species, with particular reference to the glial/non glial cell compartments. The distribution of carnosine-like immunoreactivity in the rabbit displays some differences only within the SEL, which could be linked to its arrangement and compartmentalization.

Carnosine-like immunoreactivity in the central nervous system of rats during postnatal development.

In the nervous system of adult rodents, the aminoacylhistidine dipeptides (carnosine and/or homocarnosine) have been shown to be expressed in three main populations of cells: the mature olfactory receptor neurons, a subset of glial cells, and the neuroblasts of the rostral migratory stream. The current study analyzed the distribution of these dipeptides during postnatal development within the rat brain and spinal cord focusing on their pattern of appearance in the glial cells. Double staining methods using antibodies against carnosine and some markers specific for immature (vimentin) and mature (glial fibrillary acidic protein and Rip) glial cell types were used. Glial immunostaining for the aminoacylhistidine dipeptides appears starting from postnatal day 6 and reaches the final distribution in 3-week-old animals. The occurrence of carnosine-like immunoreactivity in astrocytes lags behind that in oligodendrocytes suggesting that, as previously demonstrated by in vitro studies, oligodendrocytes are also able to synthesize carnosine and/or homocarnosine in vivo. Furthermore, the spatiotemporal patterns observed support the hypothesis that the production of these dipeptides coincides with the final stages of glia differentiation. In addition, a strong carnosine-like immunoreactivity is transiently seen in a small population of cells localized in the hypothalamus and in the subfornical organ from birth to postnatal day 21. In these cells, carnosine-like immunoreactivity was not colocalized with any of the glial specific markers used. Moreover, no evidence for colocalization of carnosine and gonadotropin-releasing hormone (GnRH) has been observed.

Carnosine-related dipeptides in neurons and glia.

Carnosine-related dipeptides have been demonstrated to occur in the nervous tissue of many vertebrates, including humans. Although several hypotheses have been formulated, to date their precise physiological role in the nervous system remains unknown. This article will review the studies on the presence and distribution of these dipeptides in the nervous system of different classes of vertebrates. It will focus on the most recent data on their cellular localization and potential functions in mammals. The studies on localization of carnosine-related dipeptides show a complex pattern of expression that involves both neuronal and glial cell types. The glial localization, widely distributed throughout the whole brain and spinal cord, includes a subset of both mature astrocytes and oligodendrocytes, whereas the neuronal localization is restricted to a particular type of neurons (the olfactory receptor neurons), and to restricted populations of putative migrating neurons and neuroblasts. There is no definitive demonstration of the function of these dipeptides in the various cell types. However, a wide array of evidence suggests that carnosine-related dipeptides could act as natural protective agents. Moreover, recent studies have suggested that, as previously postulated for the olfactory receptor neurons, in mature functional glial cells as well, carnosine-related dipeptides could be implicated in a neuromodulatory functional mechanism.

Carnosine-related dipeptides in the mammalian brain.

Carnosine and structurally related dipeptides are a group of histidine-containing molecules widely distributed in vertebrate organisms and particularly abundant in muscle and nervous tissue. Although many theories have been proposed, the biological function(s) of these compounds in the nervous system remains enigmatic. The purpose of this article is to review the distribution of carnosine-related dipeptides in the mammalian brain, with particular reference to some cell populations wherein these molecules have been demonstrated to occur very recently. The high expression of carnosine in the mammalian olfactory receptor neurons led to infer that this dipeptide could play a role as a neurotransmitter/modulator in olfaction. This prediction, which has not yet been fully demonstrated, does not explain the localization of carnosine-related dipeptides in other cell types, such as glial and ependymal cells. A recent demonstration of high carnosine-like immunoreactivity in the subependymal layer of rodents, an area of the forebrain which shares with the olfactory neuroepithelium the occurrence of continuous neurogenesis during adulthood, supports the hypothesis that carnosine-related dipeptides could be implicated in some forms of structural plasticity. However, the particular distribution of these molecules in the subependymal layer, along with their expression in glial/ependymal cell populations, suggests that they are not directly linked to cell migration or cell renewal. In the absence of a unified theory about the role of carnosine-related dipeptides in the nervous system, some common features shared by different cell populations of the mammalian brain which contain these molecules are discussed.

The subependymal layer in rodents: a site of structural plasticity and cell migration in the adult mammalian brain.

The persistence of neurogenesis and structural plasticity was believed until recently to be restricted to lower vertebrates and songbirds. Nevertheless, it has now been ascertained that these phenomena can occur in the adult mammalian nervous system, at least in three distinct sites: the olfactory neuroepithelium of the nasal mucosa and two brain regions, namely, the hippocampal dentate gyrus and the olfactory bulb. The newly generated cells of the olfactory bulb originate from the subependymal layer, a remnant of the primitive subventricular zone persisting in the adult forebrain. Besides being characterized by high rates of cell proliferation, the subependymal layer is a site of long-distance tangential cell migration, wherein migrating cells form chains enwrapped by a particular type of astrocytes. These glial cells give rise to channels (glial tubes) that separate single chains from the surrounding mature tissue. The cellular composition and the pattern of cell migration in the mammalian subependymal layer appear to be quite different in neonatal and adult animals, changing strikingly in the postnatal period. Other features of uniqueness involve the capability of neuronal precursors to divide while undergoing migration and the presence of multipotent stem cells. Thus, the subependymal layer is an area of the adult mammalian brain endowed with a cohort of phenomena proper of neural development, persisting into (and adapted to) the fully mature nervous tissue. Such features make this system an optimal model to unravel mechanisms permitting highly dynamic structural plasticity during adulthood, in the perspective of providing strategies for possible brain repair.

Carnosine-like immunoreactivity in astrocytes of the glial tubes and in newly-generated cells within the tangential part of the rostral migratory stream of rodents.

In the nervous system, the aminoacylhistidine dipeptide carnosine (beta-alanyl-L-histidine) has been shown to be expressed in the olfactory receptor neurons and in brain astrocytes. Using immunocytochemical techniques, we report here a dense carnosine-like immunoreactivity in the subependymal layer of the rodent forebrain. Since the subependymal layer involves two distinct compartments (astrocytic cells forming glial tubes and newly-generated cells of the rostral migratory stream, here organized to form chains contained within the glial tubes [Brannon Thomas L. et al. (1996) Glia 17, 1-14; Jancovski A. and Sotelo C. (1996) J. comp. Neurol. 258, 112-124; Lois C. et al (1996) Science 271, 978-981; Peretto P. et al. (1997) Brain Res. Bull. 42, 9-21]), we investigated in detail the cellular distribution of carnosine-like immunoreactivity in this area. By using double labelling techniques with antisera raised against carnosine and specific markers of glial tubes or chains of migrating cells, we show that carnosine-like immunoreactivity is associated with both the compartments. On the other hand, unlike markers of the rostral migratory stream, carnosine-like immunoreactivity was not observed in isolated, migrating cells located outside the subependymal layer, which spread through the olfactory bulb in a radially-oriented manner. This suggests that carnosine is transiently expressed by cells of the rostral migratory stream when moving in the tangentially-oriented part of the migration route. Moreover, we investigated the distribution of carnosine-like immunoreactivity in the postnatal rat forebrain and found that it is detectable in the subependymal layer only starting from the third postnatal week, although it is well known that the dipeptide is present in the olfactory receptor neurons since the embryonic day 16 [Biffo S. et al. (1992) J. chem Neuroanat. 5, 5162]. Taken together, these results show that camosine, other than abundantly present in astrocytes of the glial tubes, is associated to the tangential part of the rostral migratory stream.

Identification of the glial cell types containing carnosine-related peptides in the rat brain.

The cellular localization of carnosine-like immunoreactivity was investigated in the adult rat forebrain and in glial cell cultures obtained from newborn rat brain. Using double staining methods, we showed that in vivo carnosine-like immunoreactivity was occurring in a large number of both glial fibrillary acidic protein (GFAP)-positive astrocytes and 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNP)-positive oligodendrocytes. In vitro, the carnosine-immunoreactive staining was restricted to a subpopulation of completely differentiated oligodendrocytes, whereas no reaction was detected in immature oligodendrocytes and in astrocytes. These observations could have profound physiopathological implications considering the role suggested for carnosine and related peptides as endogenous antioxidants, free radical scavengers and anti-glycating agents of the central nervous system (CNS).

The cytosolic phosphoprotein stathmin is expressed in the olfactory system of the adult rat.

Stathmin is a cytosolic protein expressed particularly in the developing nervous system, whose phosphorylation is correlated with the action of multiple extracellular stimuli regulating cell proliferation and differentiation. In this study, we used an antibody that specifically recognizes the carboxyterminal region of stathmin to analyze the distribution of this protein in the olfactory system of adult rats, and found a high and selective immunoreactivity in immature olfactory receptors of the olfactory neuroepithelium and in cells of the rostral migratory stream. These results reveal an expression of stathmin in regions of the adult nervous system characterized by striking structural plasticity and cell renewal, suggesting that this protein could play a role in the differentiation of newly generated cell populations.

Newly-generated cells from the rostral migratory stream in the accessory olfactory bulb of the adult rat.

Cell proliferation in the accessory olfactory bulb of the adult rat was analysed after systemic injection of 5-bromo-2'-deoxyuridine, detected immunocytochemically at different survival times and compared with proliferating cell nuclear antigen immunostaining. As previously described in the main olfactory bulb, local cell proliferation was absent or very limited. By contrast, starting from 15 days after bromodeoxyuridine administration, many immunoreactive nuclei were present in the granular layer, and to a lesser extent, in other layers of the accessory olfactory bulb. This suggests that the newly-generated cells are migrating elements of the rostral migratory stream which are known to reach the olfactory bulb in 15 days. By immunocytochemical detection of the polysialylated isoform of the neural cell adhesion molecule, a weakly-adhesive cell-surface molecule expressed by newly-generated/migrating cells of the rostral migratory stream, we found a high number of immunoreactive cells in the different layers of the accessory olfactory bulb. Most of these cells were observed in the granular layer and showed the morphology of migrating neuroblasts. Some immunoreactive cells displaying neuronal morphology were also detected in the external plexiform and glomerular layers. Double labelling experiments demonstrated that these cells are newly-generated cells. These results demonstrate the occurrence of newly-added cells in the accessory olfactory bulb of the adult rat, which likely correspond to the neuronal precursors originating from the rostral migratory stream. This could be relevant since the accessory olfactory bulb of rodents plays an important role in the hard wiring of a simple olfactory memory system for sexual pheromones.

Glial tubes in the rostral migratory stream of the adult rat.

In the central nervous system cell migration is usually restricted to developmental periods and occurs mainly radially, following the radial glia. Nevertheless, in the subependymal layer of the adult rodent forebrain tangential migration of newly generated neuronal precursors directed to the olfactory bulb, which follow a well-defined pathway without dispersion, has been recently demonstrated. In the present study, by using light microscopic immunocytochemistry for glia-associated antigens (glial fibrillary acidic protein, S-100 and vimentin), and conventional electron microscopy, we observed a dense mesh-work of astrocytic cells and processes throughout the subependymal layer of the adult rat. These cells were organized to form tangentially oriented glial tubes in the subependymal layer of the lateral ventricle and in its rostral extension to the olfactory bulb. Glial tubes were particularly evident within the rostral extension and were widely intercommunicating. Using markers for the proliferating/ migrating cells of the rostral migratory stream (5-bromo-2'-deoxyuridine, PSA-NCAM, class III beta-tubulin), we provide evidence that long chains of PSA-NCAM/beta-tubulin-positive, newly generated cells are consistently observed inside the glial tubes. These results demonstrate the existence of a peculiar glial organization within the subependymal layer of the adult rat, consisting of long astrocytic tubes that likely represent a new type of glial guidance, accounting for the tangential migration of a high number of cells along their restricted pathway, to the olfactory bulb.

Development and migration of olfactory neurones in the nervous system of the neonatal opossum.

The neonatal opossum (Monodelphis domestica) was used to assess how different populations of cells are generated in the olfactory region, and how they migrate along pathways to the central nervous system. Developing nerve cells were immunocytochemically labelled using antisera directed against two specific markers of olfactory receptor neurones: olfactory marker protein (OMP) and the dipeptide carnosine. In new-born opossums both carnosine and OMP are already co-expressed in primary olfactory neurones and in those axons that extend towards the olfactory bulb. Expression of these markers in olfactory receptor neurones during the first postnatal days reflects the advanced developmental state of this system compared to other regions of the central nervous system (such as the cortex and cerebellum), which are highly immature and less developed in comparison with those of new-born rats or mice. A second, distinct population of carnosine/OMP expressing cells was also identified during the first postnatal week. These neurones were present as clusters along the olfactory nerve bundles, on the ventral-medial aspect of the olfactory bulb and in the basal prosencephalon. The distribution of this cell population was compared to another group of well characterized migratory neurones derived from the olfactory placode, which express the decapeptide GnRH (Gonadotropin-releasing hormone, also known as LHRH). GnRH was never co-localized with carnosine/OMP in the same migratory cells. These observations show that distinct cell populations arise from the olfactory placode in the neonatal opossum and that they migrate to colonize the central nervous system by following common pathways.

Cell migration from the olfactory placode and the ontogeny of the neuroendocrine compartments.

The olfactory placode and its derivative, the olfactory pit, give rise to several different populations of migrating cells, which contribute to drive the organization of the prosencephalon, but also to form a part of the central neuroendocrine compartments. Some cell types are seemingly transient and can play a role in the establishment of the final connections. The understanding of the mechanisms involved in the migration and differentiation of these cell populations can give an insight on the interplay between peripheral structures and central nervous system and on the mechanisms of commitment, phenotype selection and control for neuroendocrine cells able to selectively "colonize" the brain.