Expression of ezrin in glial tubes in the adult subventricular zone and rostral migratory stream

Ezrin is a member of the ERM (ezrin-radixin-moesin) family of membrane-cytoskeletal linking proteins. ERM proteins are involved in a wide variety of cellular functions including cell motility, signal transduction, cell-cell interaction and cell-matrix recognition. A recent in situ hybridization study showed that the mRNA encoding ezrin is expressed in neurogenic regions of the mature brain including the subventricular zone (SVZ) and rostral migratory stream (RMS); however, the specific cell types expressing ezrin and their relationship to migrating and proliferating cells in these regions have not been characterized previously. In this study, we used immunocytochemistry to perform double labeling with a variety of cell-type specific markers to characterize the expression of ezrin in the SVZ and RMS of adult mice. Ezrin was expressed at high levels in both the SVZ and RMS where ezrin-immunopositive processes formed a trabecular network surrounding the proliferating and migrating cells. Ezrin-positive cells co-labeled with the glial makers S100beta and GFAP (glial fibrillary acidic protein), but only minimally with the early neuronal markers beta III tubulin and polysialylated form of neural cell adhesion molecule 1 (PSA-NCAM), indicating that ezrin was expressed primarily in the glial tube cells. Ezrin positive cells also expressed beta-catenin, a membrane-complex protein previously implicated in the regulation of stem-cell proliferation and neuronal migration. Glial tube cells act as both precursors of, and a physical channel for, migrating neuroblasts. Bi-directional signals between glial tube cells and migrating neuroblasts have been shown to regulate the rates of both proliferation of the precursor cells and migration of the newly generated neuroblasts. Our finding that ezrin and beta-catenin are both present at the cell membrane of the glial tube cells suggests that these proteins may be involved in those signaling processes.     

Nonsynaptic GABA signaling in postnatal subventricular zone controls proliferation of GFAP-expressing progenitors

In the postnatal subventricular zone (SVZ), local cues or signaling molecules released from neuroblasts limit the proliferation of glial fibrillary acidic protein (GFAP)-expressing progenitors thought to be stem cells. However, signals between SVZ cells have not been identified. We show that depolarization of neuroblasts induces nonsynaptic SNARE-independent GABA(A) receptor currents in GFAP-expressing cells, the time course of which depends on GABA uptake in acute mouse slices. We found that GABA(A) receptors are tonically activated in GFAP-expressing cells, consistent with the presence of spontaneous depolarizations in neuroblasts that are sufficient to induce GABA release. These data demonstrate the existence of nonsynaptic GABAergic signaling between neuroblasts and GFAP-expressing cells. Furthermore, we show that GABA(A) receptor activation in GFAP-expressing cells limits their progression through the cell cycle. Thus, as GFAP-expressing cells generate neuroblasts, GABA released from neuroblasts provides a feedback mechanism to control the proliferation of GFAP-expressing progenitors by activating GABA(A) receptors.     

Immunocytochemical localization of GABA neurons and dopamine neurons in the rat main and accessory olfactory bulbs

Immunocytochemical localization of GABA neurons and dopamine neurons in the rat olfactory bulb was obtained with sheep antiserum to glutamate decarboxylase (GAD) and rabbit antiserum to tyrosine hydroxylase (TH). GAD-positive neurons include periglomerular cells, granule cells, superficial and deep short axon cells. TH-positive neurons represent periglomerular cells. Two-color immunocytochemistry shows that GABA and dopamine periglomerular cells are separate populations. The accessory olfactory bulb has rare dopamine cells and few superficial short axon cells. Radial gradients of GAD-immunostaining are evident in the main but not in the accessory olfactory bulb.     

Immunohistochemical localisation of the creatine transporter in the rat brain

Creatine (Cr) is required to maintain ATP levels in the brain. The transport of Cr across the blood–brain barrier and into neurones requires a specific creatine transporter (CRT). Mutations in the CRT gene (SLC6A8) result in a novel form of X-linked mental retardation, characterised by developmental delays, seizures and a complete absence of Cr from the brain. To identify cell types and regions that depend on Cr for energy metabolism we have determined the regional and cellular localisation of CRT protein in the rat brain using immunohistochemical techniques with a highly specific, affinity-purified, CRT antibody. The results show high levels of CRT localisation is associated with specific brain regions and certain cell types. The CRT is predominantly found in neurones. CRT immunoreactivity is particularly abundant in the olfactory bulb, granule cells of the dentate gyrus of the hippocampus, pyramidal neurones of the cerebral cortex, Purkinje cells of the cerebellum, motor and sensory cranial nerve nuclei in the brainstem and the dorsal and ventral horns of the spinal cord. Low levels of CRT were seen in the basal ganglia and white matter. Overall, CRT was found to show high intensities of labelling in the major motor and sensory regions of the forebrain, brainstem and spinal cord and forebrain regions associated with learning, memory and limbic functions. It is hypothesised that regions with high CRT expression are likely to have high metabolic ATP requirements and that areas with low CRT levels are those regions which are particularly vulnerable in neurodegenerative diseases.     

Short term treatment with estradiol decreases the rate of newly generated cells in the subventricular zone and main olfactory bulb of adult female mice

Adult neurogenesis occurs most notably in the subgranular zone (SGZ) of the hippocampal dentate gyrus and in the olfactory bulb (OB) where new neurons are generated from neural progenitors cells produced in the subventricular zone (SVZ) of the forebrain. As it is well known that gonadal steroid hormones, primarily estradiol, modulate neurogenesis in the hippocampus of adult female rodents, we wanted to determine whether estradiol would also affect the proliferation of progenitor cells in the SVZ and by consequence the rate of newly generated cells in the main OB. Thus a first group of adult female C57Bl6/J mice was ovariectomized and received a short term treatment with estradiol (single injection of 1 or 10 μg 17β-estradiol or Silastic capsule of estradiol during 2 days) before receiving a single injection with BrdU to determine whether estradiol would modulate the cell proliferation in the SVZ. A second group of adult ovariectomized female mice was submitted to the same estradiol treatment before receiving four BrdU injections, and was sacrificed 21 days later to determine whether a modulation in cell proliferation actually leads to a modulation in the number of newborn cells in the main OB. We observed a decrease in cell proliferation in the SVZ following either dose of estradiol compared to the controls. Furthermore, 21 days after their generation in the SVZ, the number of BrdU labeled cells was also lower in the main OB, both in the granular and periglomerular cell layers of estradiol-treated animals. These results show that a short term treatment with estradiol actually downregulates cell proliferation leading to a decreased number of newborn cells in the OB.     

The olfactory conditioning in the early postnatal period stimulated neural stem/progenitor cells in the subventricular zone and increased neurogenesis in the olfactory bulb of rats

The olfactory memory acquired during the early postnatal period is known to be maintained for a long period, however, its neural mechanism remains to be clarified. In the present study, we examined the effect of olfactory conditioning during the early postnatal period on neurogenesis in the olfactory bulb of rats. Using the bromodeoxyuridine-pulse chase method, we found that the olfactory conditioning, which was a paired presentation of citral odor (conditioned stimulus) and foot shock (unconditioned stimulus) in rat pups on postnatal day 11, stimulated the proliferation of neural stem/progenitor cells in the anterior subventricular zone (aSVZ), but not in the olfactory bulb, at 24 h after the conditioning. However, the number of newborn cells in the olfactory bulb was increased at 2 weeks, but not 8 weeks, after such conditioning. Neither the exposure of a citral odor alone nor foot shock alone affected the proliferation of neural stem/progenitor cells in the aSVZ at 24 h after and the number of newborn cells in the olfactory bulb at 2 weeks after. The majority of newborn cells in the olfactory bulb of either the conditioned rats or the unconditioned rats expressed the neural marker NeuN, thus indicating that the olfactory conditioning stimulated neurogenesis in the olfactory bulb. These results suggest that olfactory conditioning during the early postnatal period temporally stimulates neurogenesis in the olfactory bulb of rats.     

Peripherally injected lipopolysaccharide induces apoptosis in the subventricular zone of young adult mice

Because the subventricular zone (SVZ) constantly supplies newly generated neurons to the olfactory bulb (OB) along the rostral migratory stream (RMS) in adult brain, SVZ-RMS-OB axis has been thought to work as a unit. We previously reported that peripherally injected lipopolysaccharide (LPS) induces apoptosis in the OB in young adult mice. Therefore, this study was undertaken to examine whether peripherally injected LPS induces apoptotic cell death also in the SVZ. Two mouse strains were used: C3H/HeN and Toll-like receptor 4-mutated C3H/HeJ, and wild-type C57BL/6 and TNFR1^−/−-2^−/−, in which the genes tumor necrosis factor receptor (TNFR)1 and TNFR2 are knocked out. Immunohistochemical study and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay done on the SVZ-RMS pathway of young adult male mice showed that peripherally injected LPS switches on the apoptotic signal by cleaving pro-caspase-3, thus possibly increasing the number of cells dying from apoptosis in these areas in adult mice. Activation of the tumor necrosis factor (TNF)-α-TNFR system played a critical role in fully inducing apoptosis in this pathway. We suggest that TNF-α was probably released not from microglia but from astrocytes in the SVZ and RMS.     

The age-dependent change in Olfactory periglomerular neuronal populations is not affected by interrupting subventricular neuroblast migration in adult rats

The olfactory bulb (OB) is rich in the number and variety of neurotransmitter and neuropeptide containing cells, in particular in the glomerular layer. Several reports suggest that numbers of some periglomerular phenotypes could change depending on age. However, it is unclear whether the different classes of periglomerular interneurons are modified or are maintained stable throughout life. Thus, our first objective was to obtain the absolute number of cells belonging to the different periglomerular phenotypes at adulthood. On the other hand, the olfactory bulb is continously supplied with newly generated periglomerular neurons produced by stem cells located in the subventricular zone (SVZ) and rostral migratory stream. Previously, we demonstrated that the implantation of a physical barrier completely prevents SVZ neuroblast migration towards the OB. Then, another objective of this study was to evaluate whether stopping the continuous supply of SVZ neuroblasts modified the different periglomerular populations throughout time. In summary, we estimated the total number of TH-IR, CalB-IR, CalR-IR and GAD-IR cells in the OB glomerular layer at several time points in control and barrier implanted adult rats. In addition, we estimated the volume of glomerular, granular and complete OB. Our main finding was that the number of the four main periglomerular populations is age-dependent, even after impairment of subventricular neuroblast migration. Furthermore, we established that these changes do not correlate with changes in the volume of glomerular layer.     

Area-specific morphological and neurochemical maturation of non-pyramidal neurons in the rat hippocampus as revealed by parvalbumin immunocytochemistry

Summary The time course of the morphological differentiation of non-pyramidal neurons in the rat hippocampus shows an area specificity. Thus, non-pyramidal neurons in CA3 appear more mature than in CA1 at early postnatal stages. Physiological data provide evidence for an earlier maturation of GABA-mediated inhibition in CA3 in comparison to CA1. As the calcium-binding protein parvalbumin (PARV) is thought to be a marker for highly active inhibitory neurons, we analyzed the area-specific appearance of PARV in GABAergic neurons during development. Employing combined light and electron microscopic immunocytochemistry, we revealed an area specificity in the time course of the neurochemical and morphological maturation of this functionally important subpopulation of non-pyramidal cells. The first appearance of PARV-immunoreactivity was observed at P7 and was exclusively located in cell bodies in CA3. At P8, neurons in CA3 exhibited PARV-immunoreactivity in cell bodies and dendrites, but very rarely in axon terminals. These neurons displayed the typical light and electron microscopic characteristics of GABAergic non-pyramidal cells. At P10, axon terminals formed typical baskets surrounding the pyramidal cells. The appearance of PARV-immunoreactivity in cell bodies, dendrites and axon terminals in CAq was noticed about 1 to 2 days later. In the fascia dentata, non-granule cells displayed immunoreactivity not before P10. These data indicate a sequential neurochemical and morphological maturation of non-pyramidal neurons that may be related to differences in the maturation of inhibition during hippocampal development.In partial fulfilment of the requirements for the degree of Dr. med. at the University of Frankfurt/Main     

TRIP6 regulates neural stem cell maintenance in the postnatal mammalian subventricular zone

Background: Postnatal neurogenesis persists throughout life in the subventricular zone (SVZ)‐olfactory bulb pathway in mammals. Extrinsic or intrinsic factors have been revealed to regulate neural stem cell (NSC) properties and neurogenesis. Thyroid hormone receptor interacting protein 6 (TRIP6) belongs to zyxin family of LIM proteins, which have been shown to interact with various proteins to mediate cellular functions. However, the role of TRIP6 in NSCs is still unknown. Results: By performing double immunofluorescence staining, we found that TRIP6 was expressed by Sox2‐positive NSCs in embryonic and postnatal mouse forebrains. To study the function of TRIP6 in NSCs, we performed overexpression and knockdown experiments with neurospheres derived from postnatal day 7 SVZ. We found that TRIP6 was necessary and sufficient for self‐renewal and proliferation of NSCs, but inhibited their differentiation. To further investigate the mechanism of TRIP6 in NSCs, we performed Luciferase reporter assay and found that TRIP6 activated Notch signaling, a pathway required for NSC self‐renewal. Conclusions: Our data suggest that TRIP6 regulates NSC maintenance and it may be a new marker for NSCs. Developmental Dynamics 243:1130–1142, 2014. © 2014 Wiley Periodicals, Inc.     

Wnt expression in the adult rat subventricular zone after stroke

Neurogenesis occurs in adult brain neural progenitor cells (NPCs) located in the subventricular zone (SVZ) of the lateral ventricle and the subgrandular zone of the hippocampal dentate gyrus. After ischemic stroke, NPCs in the SVZ proliferate and migrate towards the ischemic boundary region to replenish damaged neurons. During development, the Wnt pathways contribute to stem cell maintenance and promote neurogenesis. We hypothesized that stroke up regulates Wnt family genes in SVZ cells. Non-ischemic and ischemic cultured SVZ cells and a single population of non-ischemic and ischemic SVZ cells isolated by laser capture microdissection (LCM) were analyzed for Wnt pathway expression using real-time RT-PCR and immunostaining. The number of neurospheres increased significantly (p<0.05) in SVZ cells derived from ischemic (32+/-4.7/rat) compared with the number in non-ischemic SVZ cells (18+/-3/rat). Wnt family gene mRNA levels were detected in SVZ cells isolated from both cultured and LCM SVZ cells, however there was no up regulation between non-ischemic and ischemic SVZ cells. Immunostaining on brain sections also demonstrated no up regulation of Wnt pathway protein between ischemic and non-ischemic SVZ cells. Expression of the Wnt family genes in SVZ cells support the hypothesis that the Wnt pathway may be involved in neurogenesis in the adult brain. However, ischemia does not up regulate Wnt family gene expression.     

Patterns of expression of the immediate-early gene egr-1 in the accessory olfactory bulb of female mice exposed to pheromonal constituents of male urine

Male mice excrete large quantities of major urinary proteins that have been proposed to have an important pheromonal role either alone or by way of their bound ligands. We have found that these major urinary proteins are not only likely to mediate the pregnancy blocking effects of male urine, but that they also convey the strain recognition signal of the male pheromone. Recent molecular biological investigations have characterized two classes of pheromonal receptor in the vomeronasal organ that appear to project separately to anterior and posterior regions of the accessory olfactory bulb. However, it is not known whether these separate pathways handle fundamentally different types of pheromonal information. We have attempted to investigate this question using the expression of the immediate-early gene egr-1 as a marker for activity of neurons in the accessory olfactory bulb of female mice in response to putative pheromonal constituents. Exposure to 2,3 dihydro-exo-brevicomin and 2-sec-butyl-4,5-dihydro-thiazole, the main ligands bound to the major urinary proteins, elicited expression of egr-1 in clusters of presumed mitral neurons at the medial and lateral margins of the posterior accessory olfactory bulb. Whole male urine and a preparation of major urinary proteins that had been stripped of their ligands induced egr-1 expression in mitral cells of the anterior half of the accessory olfactory bulb in addition to the posterior clusters.

This would suggest that the anterior and posterior halves of the accessory olfactory bulb are processing different aspects of the male pheromone signal with the anterior region, which responds preferentially to major urinary proteins, being principally concerned with the strain recognition component.     

GABAergic basal forebrain afferents innervate selectively GABAergic targets in the main olfactory bulb

In this work we have analyzed the targets of the GABAergic afferents to the main olfactory bulb originating in the basal forebrain of the rat. We combined anterograde tracing of 10 kD biotinylated dextran amine (BDA) injected in the region of the horizontal limb of the diagonal band of Broca that projects to the main olfactory bulb, with immunocytochemical detection of GABA under electron microscopy or vesicular GABA transporter (vGABAt) under confocal fluorescent microscopy. GABAergic afferents were identified as double labeled BDA-GABA boutons. Their targets were identified by their ultrastructure and GABA content. We found that GABAergic afferents from the basal forebrain were distributed all over the bulbar lamination, but were more abundant in the glomerular and inframitral layers (i.e. internal plexiform layer and granule cell layer). The fibers had thick varicosities with abundant mitochondria and large perforated synaptic specializations. They contacted exclusively GABAergic cells, corresponding to type 1 periglomerular cells in the glomerular layer, and to granule cells in inframitral layers. This innervation will synchronize the bulbar inhibition and consequently the response of the principal cells to the olfactory input. The effect of the activation of this pathway will produce a disinhibition of the bulbar principal cells. This facilitation might occur at two separate levels: first in the terminal tufts of mitral and tufted cells via inhibition of type 1 periglomerular cells; second at the level of the firing of the principal cells via inhibition of granule cells. The GABAergic projection from the basal forebrain ends selectively on interneurons, specifically on type 1 periglomerular cells and granule cells, and is likely to control the activity of the olfactory bulb via disinhibition of principal cells. Possible similarities of this pathway with the septo-hippocampal loop are discussed.     

Biophysical properties and ionic signature of neuronal progenitors of the postnatal subventricular zone in situ

Previous studies have reported the presence of neuronal progenitors in the subventricular zone (SVZ) and rostral migratory stream (RMS) of the postnatal mammalian brain. Although many studies have examined the survival and migration of progenitors after transplantation and the factors influencing their proliferation or differentiation, no information is available on the electrophysiological properties of these progenitors in a near-intact environment. Thus we performed whole cell and cell-attached patch-clamp recordings of progenitors in brain slices containing either the SVZ or the RMS from postnatal day 15 to day 25 mice. Both regions displayed strong immunoreactivity for nestin and neuron-specific class III beta-tubulin, and recorded cells displayed a morphology typical of the neuronal progenitors known to migrate throughout the SVZ and RMS to the olfactory bulb. Recorded progenitors had depolarized zero-current resting potentials (mean more depolarized than -28 mV), very high input resistances (about 4 GOmega), and lacked action potentials. Using the reversal potential of K+ currents through a cell-attached patch a mean resting potential of -59 mV was estimated. Recorded progenitors displayed Ca2+-dependent K+ currents and TEA-sensitive-delayed rectifying K+ (KDR) currents, but lacked inward K+ currents and transient outward K+ currents. KDR currents displayed classical kinetics and were also sensitive to 4-aminopyridine and alpha-dendrotoxin, a blocker of Kv1 channels. Na+ currents were found in about 60% of the SVZ neuronal progenitors. No developmental changes were observed in the passive membrane properties and current profile of neuronal progenitors. Together these data suggest that SVZ neuronal progenitors display passive membrane properties and an ionic signature distinct from that of cultured SVZ neuronal progenitors and mature neurons.     

A removable head-mounted microdrive for unit recording in the free-behaving rat

The microdrive described in this paper has been used for two years for chronic unit recording in the olfactory bulbs of unrestrained rats. It is a removable micromanipulator, suitable for tungsten microelectrodes, designed for stereotaxic exploration of cylindrical areas of nervous tissue 9 mm high and 1.6 mm in dia. The electrode is moved up and down without any rotation and with minimal vibration, with a degree of precision of 5 microns.     

Olfactory bulb ventricles as a frequent finding in magnetic resonance imaging studies of the olfactory system

Background. In some species an embryologic cavity inside the olfactory bulb (OB) persists and is called olfactory bulb ventricle (OBV). It is generally assumed that OBVs in humans are solitary findings representing remnants of embryologic structures that were not fully regressed, although the incidence of OBVs was never examined. Using magnetic resonance imaging (MRI), the present study aimed to study the incidence of OBVs in healthy human subjects. Material and methods. A total of 122 individuals participated. Volumes of the right and left OB were determined using MRI scans and a standardized protocol for OB analysis. For comparison, OBs of 42 cadavers were collected and sectioned. Results. The main finding of this study was the high incidence of OBV-like structures in our study group. Seventy-two out of 122 (59%) participants yielded signs for an OBV whereas three out of 42 postmortem OBs contained histologically detectable OBV. Discussion: This stands in disagreement with the previous assumption of complete obliteration at the time of birth. This discrepancy may be explained by the fact that our present findings are based on modern MRI techniques with much higher resolution than 10 or 20 years ago. Another possible explanation for the discrepancy between studies based on MRI and histopathology might relate to postmortem resorption of cerebrospinal fluid from OBVs. Especially with a long postmortem interval OBVs may collapse and may no longer appear as an open cavity.