The Ginkgo biloba extract modulates the balance between proliferation and differentiation in the olfactory epithelium of adult mice following bulbectomy.

Abstract - The adult olfactory receptor neurons (ORNs), located in the olfactory epithelium (OE) are permanently renewed thanks to neuronal progenitors present in the deep part of the OE, the globose basal cells (GBCs). Following the ablation of their synaptic target, the olfactory bulb (OB), ORNs degenerate by apoptosis and a wave of neurogenesis, including proliferation of GBCs and neuronal differentiation of their progeny, restores the olfactory function. The Ginkgo biloba extract (EGb 761) (Beaufour Ipsen, France) was administered to adult mice at the doses of 50 or 100 mg/kg, following bilateral bulbectomy and its effects on the expression of PCNA, reflecting the number of proliferating GBCs and on growth associated protein 43 (GAP-43), expressed by differentiating neurons were measured by Western blotting. PCNA expression peaked 9 days post-bulbectomy in untreated animals, but 7 days post-lesion in EGb 761-treated animals. A simultaneous reduction in GAP-43 expression suggested that EGb 761 may temporarily favor the proliferation of GBCs rather than their entry into the differentiation pathway. Probably as a consequence of the earlier onset of the neurogenetic response to bulbectomy, neuronal differentiation was enhanced in the OE, 3 weeks post-bulbectomy. These data suggest that EGb 761 may have beneficial effects upon neurogenesis in the OE through changing the balance between proliferation and differentiation.     

Neurogenesis and cell death in olfactory epithelium

The olfactory epithelium (OE) of the mammal is uniquely suited as a model system for studying how neurogenesis and cell death interact to regulate neuron number during development and regeneration. To identify factors regulating neurogenesis and neuronal death in the OE, and to determine the mechanisms by which these factors act, investigators studied OE using two major experimental paradigms: tissue culture of OE; and ablation of the olfactory bulb or severing the olfactory nerve in adult animals, procedures that induce cell death and a subsequent surge of neurogenesis in the OE in vivo. These studies characterized the cellular stages in the olfactory receptor neuron (ORN) lineage, leading to the realization that at least three distinct stages of proliferating neuronal precursor cells are employed in generating ORNs. The identification of a number of factors that act to regulate proliferation and survival of ORNs and their precursors suggests that these multiple developmental stages may serve as control points at which cell number is regulated by extrinsic factors. In vivo surgical studies, which have shown that all cell types in the neuronal lineage of the OE undergo apoptotic cell death, support this idea. These studies, and the possible coregulation of neuronal birth and apoptosis in the OE, are discussed. © 1996 John Wiley & Sons, Inc.     

Decreased Hippocampal Neurogenesis Following Olfactory Bulbectomy is Reversed by Repeated Citalopram Administration

1. Whereas much progress has been made in the treatment of depression, the exact pathogenetic mechanisms of the disorder are still poorly understood. It has been proposed that one possible mechanism could be a decrease in adult hippocampal neurogenesis.2. The olfactory bulbectomy (OB) in rats is widely accepted as an animal model of depression. In the present study, we investigated whether hippocampal neurogenesis is affected by an OB, and whether chronic citalopram, a serotonin selective reuptake inhibitor, counteracts OB-induced impairment of neurogenesis.3. Our study shows that OB decreases proliferation of the neuronal precursors in the dentate gyrus and retards their differentiation into mature granule neurons. In OB rats, repeated administration of citalopram restores reduced proliferative activity and enhances the differentiation of precursors into mature calbindin-positive neurons.4. The obtained data demonstrate that a citalopram-induced increase in neurogenesis in OB rats could be one possible mechanism by which antidepressants alleviate OB-induced depressive-like behavior.     

Olfactory epithelia differentially express neuronal markers

All three olfactory epithelia, the olfactory epithelium proper (OE), the septal organ of Masera (SO), and the vomeronasal organ of Jacobson (VNO) originate from the olfactory placode. Here, their diverse neurochemical phenotypes were analyzed using the immunohistochemical expression pattern of different neuronal markers. The olfactory bulb (OB) served as neuronal control. Neuronal Nuclei Marker (NeuN) is neither expressed in sensory neurons in any of the three olfactory epithelia, nor in relay neurons (mitral/tufted cells) of the OB. However, OB interneurons (periglomerular/granule cells) labeled, as did supranuclear structures of VNO supporting cells and VNO glands. Protein Gene Product 9.5 (PGP9.5 = C-terminal ubiquitin hydrolase L1 = UCHL1) expression is exactly the opposite: all olfactory sensory neurons express PGP9.5 as do OB mitral/tufted cells but not interneurons. Neuron Specific Enolase (NSE) expression is highest in the most apically located OE and SO sensory neurons and patchy in VNO. In contrast, the cytoplasm of the most basally located neurons of OE and SO immunoreacted for Growth Associated Protein 43 (GAP-43/B50). In VNO neurons GAP-43 labeling is also nuclear. In the cytoplasm, Olfactory Marker Protein (OMP) is most intensely expressed in SO, followed by OE and least in VNO neurons; further, OMP is also expressed in the nucleus of basally located VNO neurons. OB mitral/tufted cells express OMP at low levels. Neurons closer to respiratory epithelium often expressed a higher level of neuronal markers, suggesting a role of those markers for neuronal protection against take-over. Within the VNO the neurons show clear apical–basal expression diversity, as they do for factors of the signal transduction cascade. Overall, expression patterns of the investigated neuronal markers suggest that OE and SO are more similar to each other than to VNO.     

Diazepam binding inhibitor promotes progenitor proliferation in the postnatal SVZ by reducing GABA signaling

The subventricular zone (SVZ) of the lateral ventricles is the largest neurogenic niche of the postnatal brain. New SVZ-generated neurons migrate via the rostral migratory stream to the olfactory bulb (OB) where they functionally integrate into preexisting neuronal circuits. Nonsynaptic GABA signaling was previously shown to inhibit SVZ-derived neurogenesis. Here we identify the endogenous protein diazepam binding inhibitor (DBI) as a positive modulator of SVZ postnatal neurogenesis by regulating GABA activity in transit-amplifying cells. We performed DBI loss- and gain-of-function experiments in vivo at the peak of postnatal OB neuron generation in mice and demonstrate that DBI enhances proliferation by preventing SVZ progenitors to exit the cell cycle. Furthermore, we provide evidence that DBI exerts its effect on SVZ progenitors via its octadecaneuropeptide proteolytic product (ODN) by inhibiting GABA-induced currents. Together our data reveal a regulatory mechanism by which DBI counteracts the inhibitory effect of nonsynaptic GABA signaling on subventricular neuronal proliferation.     

Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors

The olfactory epithelium (OE) contains neural precursor cells which can be easily harvested from a minimally invasive nasal biopsy, making them a valuable cell source to study human neural cell lineages in health and disease. Glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology and treatment of neuropsychiatric disorders and also in the regulation of murine neural precursor cell fate in vitro and in vivo. In this study, we examined the impact of decreased GSK-3 activity on the fate of adult human OE neural precursors in vitro. GSK-3 inhibition was achieved using ATP-competitive (6-bromoindirubin-3′-oxime and CHIR99021) or substrate-competitive (TAT-eIF2B) inhibitors to eliminate potential confounding effects on cell fate due to off-target kinase inhibition. GSK-3 inhibitors decreased the number of neural precursor cells in OE cell cultures through a reduction in proliferation. Decreased proliferation was not associated with a reduction in cell survival but was accompanied by a reduction in nestin expression and a substantial increase in the expression of the neuronal differentiation markers MAP1B and neurofilament (NF-M) after 10 days in culture. Taken together, these results suggest that GSK-3 inhibition promotes the early stages of neuronal differentiation in cultures of adult human neural precursors and provide insights into the mechanisms by which alterations in GSK-3 signaling affect adult human neurogenesis, a cellular process strongly suspected to play a role in the etiology of neuropsychiatric disorders.     

Protection of DAergic neurons mediates treadmill running attenuated olfactory deficits and olfactory neurogenesis promotion in depression model

In this study, we aimed to test the effects of treadmill running on depression induced olfactory functions and OB neurogenesis in depression model. Depression model was created with chronic unpredictable mild stress (CUMS) and treadmill running was performed as the antidepressant treatment. Behavioral results showed that treadmill running not only attenuated the depression mood but also improved the olfactory discrimination and sensitivity in CUMS depression model. Immune-staining further indicates treadmill running promoted neurogenesis in hippocampal OB region. Moreover, treadmill running prevented the loss of DAergic neurons in glomerular layer of OB region, indicating the critical role of DAergic neuronal functions in regulating treadmill running mediated olfactory functions. In depression model, inhibiting DAergic neurons by intra-OB injection of 6-OHDA resulted in the compromised improving effects of treadmill running olfactory discrimination. In conclusion, treadmill running could attenuate depression associated olfactory deficits by promoting olfactory neurogenesis and improve DAergic neural functions.     

Six1 is essential for early neurogenesis in the development of olfactory epithelium

The olfactory epithelium (OE) is derived from the olfactory placode (OP) during mouse development. At embryonic day (E) 10.0-E10.5, “early neurogenesis” occurs in the OE, which includes production of pioneer neurons that emigrate out of the OE and other early-differentiated neurons. Around E12.5, the OE becomes organized into mature pseudostratified epithelium and shows “established neurogenesis,” in which olfactory receptor neurons (ORNs) are differentiated from basal progenitors. Little is known about the molecular pathway of early neurogenesis. The homeodomain protein Six1 is expressed in all OP cells and neurogenic precursors in the OE. Here we show that early neurogenesis is severely disturbed despite the unaltered expression of Mash1 at E10.5 in the Six1-deficient mice (Six1^−/−). Expression levels of neurogenin1 (Ngn1) and NeuroD are reduced and those of Hes1 and Hes5 are augmented in the OE of Six1^−/− at E10.5. Pioneer neurons and cellular aggregates, which are derived from the OP/OE and situated in the mesenchyme between the OE and forebrain, are completely absent in Six1^−/−. Moreover, ORN axons and the gonadotropin-releasing hormone-positive neurons fail to extend and migrate to the forebrain, respectively. Our study indicates that Six1 plays critical roles in early neurogenesis by regulating Ngn1, NeuroD, Hes1, and Hes5.     

Glycoconjugates are stage- and position-specific cell surface molecules in the developing olfactory system, 2: Unique carbohydrate antigens are topographic markers for selective projection patterns of olfactory axons

Three monoclonal antibodies specific for different carbohydrate antigens were used to analyze the development of the olfactory system in rats. CC2 antibodies react with a subset of main olfactory neurons, their axons, and terminals in the olfactory bulb. CC2 antigens are expressed on dorsomedial neurons in the olfactory epithelium (OE) from embryonic (E) day 15 to adults. In the olfactory bulb (OB), only dorsomedially located glomeruli express CC2 glycoconjugates from postnatal day (P) 2 to adults. Thus CC2 defines a dorsomedially organized projection that is established early in embryonic development and continues in adults. P-Path antibodies react with antigens that are expressed on the olfactory nerve in embryos, and are also detected on cell bodies in the neuroepithelium and in glomeruli of the OB at P2. At P14, P-Path staining is weaker, but remains present on many cells in the epithelium and in many glomeruli in the bulb. Postnatally, P-Path immunostaining continues to decrease in most regions of the OE and OB. At P35 and afterwards, only a few P-Path-positive neuronal cells can be detected in the OE. Furthermore, after P35 only two groups of glomeruli in the OB are P-Path immunoreactive. One is situated adjacent to the accessory olfactory bulb (AOB) at the dorsocaudal surface of the OB. The other is adjacent to the AOB at the ventrocaudal surface of the OB. Thus, in adults, P-Path glycoconjugates are expressed in neurons and axons that project only to a specific subset of caudal glomeruli of the OB. Monoclonal antibody 1B2, reacts with β-galactose-terminating glycolipids and glycoproteins. At P2, 1B2 immunoreactivity is seen on a subset of cell bodies that are distributed throughout the OE and is expressed in most glomeruli in the OB at this age. By P35 and in adults, 1B2 continues to be expressed on a subset of neurons in the OE that project to only a small subset of glomeruli in the OB. Unlike CC2 and P-Path antigens that define specific groups of glomeruli, 1B2-immunoreactive glomeruli do not have a detectable spatial pattern. It is more likely that 1B2 antigens define a specific stage in the maturation of connections between the OE and OB.     

The critical role of cyclin D2 in adult neurogenesis

Adult neurogenesis (i.e., proliferation and differentiation of neuronal precursors in the adult brain) is responsible for adding new neurons in the dentate gyrus of the hippocampus and in the olfactory bulb. We describe herein that adult mice mutated in the cell cycle regulatory gene Ccnd2, encoding cyclin D2, lack newly born neurons in both of these brain structures. In contrast, genetic ablation of cyclin D1 does not affect adult neurogenesis. Furthermore, we show that cyclin D2 is the only D-type cyclin (out of D1, D2, and D3) expressed in dividing cells derived from neuronal precursors present in the adult hippocampus. In contrast, all three cyclin D mRNAs are present in the cultures derived from 5-day-old hippocampi, when developmental neurogenesis in the dentate gyrus takes place. Thus, our results reveal the existence of molecular mechanisms discriminating adult versus developmental neurogeneses.     

Glycoconjugates are stage- and position-specific cell surface molecules in the developing olfactory system, 2: Unique carbohydrate antigens are topographic markers for selective projection patterns of olfactory axons.

Three monoclonal antibodies specific for different carbohydrate antigens were used to analyze the development of the olfactory system in rats. CC2 antibodies react with a subset of main olfactory neurons, their axons, and terminals in the olfactory bulb. CC2 antigens are expressed on dorsomedial neurons in the olfactory epithelium (OE) from embryonic (E) day 15 to adults. In the olfactory bulb (OB), only dorsomedially located glomeruli express CC2 glycoconjugates from postnatal day (P) 2 to adults. Thus CC2 defines a dorsomedially organized projection that is established early in embryonic development and continues in adults. P-Path antibodies react with antigens that are expressed on the olfactory nerve in embryos, and are also detected on cell bodies in the neuroepithelium and in glomeruli of the OB at P2. At P14, P-Path staining is weaker, but remains present on many cells in the epithelium and in many glomeruli in the bulb. Postnatally, P-Path immunostaining continues to decrease in most regions of the OE and OB. At P35 and afterwards, only a few P-Path-positive neuronal cells can be detected in the OE. Furthermore, after P35 only two groups of glomeruli in the OB are P-Path immunoreactive. One is situated adjacent to the accessory olfactory bulb (AOB) at the dorsocaudal surface of the OB. The other is adjacent to the AOB at the ventrocaudal surface of the OB. Thus, in adults, P-Path glycoconjugates are expressed in neurons and axons that project only to a specific subset of caudal glomeruli of the OB. Monoclonal antibody 1B2, reacts with beta-galactose-terminating glycolipids and glycoproteins. At P2, 1B2 immunoreactivity is seen on a subset of cell bodies that are distributed throughout the OE and is expressed in most glomeruli in the OB at this age. By P35 and in adults, 1B2 continues to be expressed on a subset of neurons in the OE that project to only a small subset of glomeruli in the OB. Unlike CC2 and P-Path antigens that define specific groups of glomeruli, 1B2-immunoreactive glomeruli do not have a detectable spatial pattern. It is more likely that 1B2 antigens define a specific stage in the maturation of connections between the OE and OB.     

Notch1 expression and ligand interactions in progenitor cells of the mouse olfactory epithelium

Despite the relatively simplified organization of the olfactory epithelium (OE), our understanding of the factors that regulate its cellular diversity is limited. Genetic and localization studies suggest that Notch signaling may be important in this process. We characterize here a population of Notch1 ⁺ olfactory basal cells in embryonic mice that coordinately express both the Notch effector Hes5 and the glycosyltransferase Lfng. These cells are distinct from Mash1 ⁺ neuronal precursors, but give rise to sensory neurons, suggesting that Notch1 signals may in part function to maintain a neurogenic progenitor pool. Furthermore, Lfng ⁺ cells also generate a population of cells in the migratory mass that appear to be ensheathing glial precursors, indicating potential multipotency in these progenitors. The Notch ligand Dll4 is expressed by basal OE cells that are interspersed with Notch1 ⁺ progenitors during later OE neurogenesis. In contrast, mice deficient in Dll1 exhibit a smaller OE and a loss of Hes5 expression, indicating an earlier function in olfactory progenitor cell development. Taken together, these results further support a role for Notch signaling in the regulation of olfactory neurogenesis and cell diversity.     

Olfactory cell derivation and migration

This special issue of the Journal of Molecular Histology is devoted to the unique phenomena of migration and adult neurogenesis observed in the olfactory system. Neural progenitors migrate from the olfactory placode and epithelium (OE) into the central nervous system (CNS) and from the forebrain ventricular region to the olfactory bulb (OB). Not unexpectedly, there are a number of controversies with regard to the mechanisms regulating these phenomena in both developing and adult animals. One especially controversial issue common to both the peripheral (OE) and central (OB) systems is the identity of the slowly dividing multipotent stem cell and the mechanisms regulating the lineage specification of these progenitors which eventually differentiate into neurons and glia. Nine contributions from leading laboratories address these and other issues with respect to progenitors and their integration into OE and OB circuitry in several species.     

Nitric oxide regulates neurogenesis in adult olfactory epithelium in vitro

Nitric oxide regulates neurogenesis in the developing and adult brain. The olfactory epithelium is a site of neurogenesis in the adult and previous studies suggest a role for nitric oxide in this tissue during development. We investigated whether neuronal precursor proliferation and differentiation is regulated by nitric oxide using primary cultures of olfactory epithelial cells and an immortalized, clonal, neuronal precursor cell line derived from adult olfactory epithelium. In these cultures NOS inhibition reduced cell proliferation and stimulated neuronal differentiation, including expression of a voltage-dependent potassium conductance of the delayed rectifier type. In the neuronal precursor cell line, differentiation was associated with a significant decrease in nitric oxide release. In contrast, addition of nitric oxide stimulated proliferation and reduced neuronal differentiation. Nitric oxide regulated olfactory neurogenesis independently of added growth factors. Taken together these results indicate that nitric oxide levels can regulate cell proliferation and neuronal differentiation of olfactory precursor cells.     

Once and again: retinoic acid signaling in the developing and regenerating olfactory pathway

Retinoic acid (RA), a member of the steroid/thyroid superfamily of signaling molecules, is an essential regulator of morphogenesis, differentiation, and regeneration in the mammalian olfactory pathway. RA-mediated teratogenesis dramatically alters olfactory pathway development, presumably by disrupting retinoid-mediated inductive signaling that influences initial olfactory epithelium (OE) and bulb (OB) morphogenesis. Subsequently, RA modulates the genesis, growth, or stability of subsets of OE cells and OB interneurons. RA receptors, cofactors, and synthetic enzymes are expressed in the OE, OB, and anterior subventricular zone (SVZ), the site of neural precursors that generate new OB interneurons throughout adulthood. Their expression apparently accommodates RA signaling in OE cells, OB interneurons, and slowly dividing SVZ neural precursors. Deficiency of vitamin A, the dietary metabolic RA precursor, leads to cytological changes in the OE, as well as olfactory sensory deficits. Vitamin A therapy in animals with olfactory system damage can accelerate functional recovery. RA-related pathology as well as its potential therapeutic activity may reflect endogenous retinoid regulation of neuronal differentiation, stability, or regeneration in the olfactory pathway from embryogenesis through adulthood. These influences may be in register with retinoid effects on immune responses, metabolism, and modulation of food intake.     

Prenatal Stress Inhibits Hippocampal Neurogenesis but Spares Olfactory Bulb Neurogenesis

The dentate gyrus (DG) and the olfactory bulb (OB) are two regions of the adult brain in which new neurons are integrated daily in the existing networks. It is clearly established that these newborn neurons are implicated in specific functions sustained by these regions and that different factors can influence neurogenesis in both structures. Among these, life events, particularly occurring during early life, were shown to profoundly affect adult hippocampal neurogenesis and its associated functions like spatial learning, but data regarding their impact on adult bulbar neurogenesis are lacking. We hypothesized that prenatal stress could interfere with the development of the olfactory system, which takes place during the prenatal period, leading to alterations in adult bulbar neurogenesis and in olfactory capacities. To test this hypothesis we exposed pregnant C57Bl/6J mice to gestational restraint stress and evaluated behavioral and anatomic consequences in adult male offspring.We report that prenatal stress has no impact on adult bulbar neurogenesis, and does not alter olfactory functions in adult male mice. However, it decreases cell proliferation and neurogenesis in the DG of the hippocampus, thus confirming previous reports on rats. Altogether our data support a selective and cross-species long-term impact of prenatal stress on neurogenesis.     

Initiation of olfactory placode development and neurogenesis is blocked in mice lacking both Six1 and Six4

Mouse olfactory epithelium (OE) originates from ectodermally derived placode, the olfactory placode that arises at the anterior end of the neural plate. Tissue grafting and recombination experiments suggest that the placode is derived from a common preplacodal domain around the neural plate and its development is directed by signals arising from the underlying mesoderm and adjacent neuroectoderm. In mice, loss of Six1 affects OE morphogenesis but not placode formation. We show here that embryos lacking both Six1 and Six4 failed to form the olfactory placode but the preplacodal region appeared to be specified as judged by the expression of Eya2, which marks the common preplacodal domain, suggesting a synergistic requirement of Six1 and Six4 in patterning the preplacodal ectoderm to a morphologic placode. Our results show that Six1 and Six4 are coexpressed in the preplacodal ectoderm from E8.0. In the olfactory pit, Six4 expression was observed in the peripheral precursors that overlap with Mash1-expressing cells, the early committed neuronal lineage. In contrast, Six1 is highly distributed in the peripheral regions where stem cells reside at E10.5 and it overlaps with Sox2 expression. Both genes are expressed in the basal and apical neuronal progenitors in the OE. Analyses of Six1;Six4 double mutant embryos demonstrated that the slightly thickened epithelium observed in the mutant was not induced for neuronal development. In contrast, in Six1^−/− embryos, all neuronal lineage markers were initially expressed but the pattern of their expression was altered. Although very few, the pioneer neurons were initially present in the Six1 mutant OE. However, neurogenesis ceased by E12.5 due to markedly increased cell apoptosis and reduced proliferation, thus defining the cellular defects occurring in Six1^−/− OE that have not been previously observed. Our findings demonstrate that Six1/4 function at the top of early events controlling olfactory placode formation and neuronal development. Our analyses show that the threshold of Six1/4 may be crucial for the expression of olfactory specific genes and that Six1 and Six4 may act synergistically to mediate olfactory placode specification and patterning through Fgf and Bmp signaling pathways.