Reduced proliferation in the adult mouse subventricular zone increases survival of olfactory bulb interneurons

Neurogenesis in the adult brain is largely restricted to the subependymal zone (SVZ) of the lateral ventricle, olfactory bulb (OB) and the dentate subgranular zone, and survival of adult-born cells in the OB is influenced by factors including sensory experience. We examined, in mice, whether survival of adult-born cells is also regulated by the rate of precursor proliferation in the SVZ. Precursor proliferation was decreased by depleting the SVZ of dopamine after lesioning dopamine neurons in the substantia nigra compacta with 6-hydroxydopamine. Subsequently, we examined the effect of reduced SVZ proliferation on the generation, migration and survival of neuroblasts and mature adult-born cells in the SVZ, rostral migratory stream (RMS) and OB. Proliferating cells in the SVZ, measured by 5-bromo-2-deoxyuridine (BrdU) injected 2 hours prior to death or by immunoreactivity against Ki67, were reduced by 47% or 36%, respectively, 7 days after dopamine depletion, and by 29% or 31% 42 days after dopamine depletion, compared to sham-treated animals. Neuroblast generation in the SVZ and their migration along the RMS were not affected, neither 7 nor 42 days after the 6-hydroxydopamine injection, since the number of doublecortin-immunoreactive neuroblasts in the SVZ and RMS, as well as the number of neuronal nuclei-immunoreactive cells in the OB, were stable compared to control. However, survival analysis 15 days after 6-hydroxydopamine and 6 days after BrdU injections showed that the number of BrdU+ cells in the SVZ was 70% higher. Also, 42 days after 6-hydroxydopamine and 30 days after BrdU injections, we found an 82% increase in co-labeled BrdU+/γ-aminobutyric acid-immunoreactive cell bodies in the granular cell layer, while double-labeled BrdU+/tyrosine hydroxylase-immunoreactive cell bodies in the glomerular layer increased by 148%. We conclude that the number of OB interneurons following reduced SVZ proliferation is maintained through an increased survival of adult-born precursor cells, neuroblasts and interneurons.     

Longterm quiescent cells in the aged human subventricular neurogenic system specifically express GFAP‐δ

A main neurogenic niche in the adult human brain is the subventricular zone (SVZ). Recent data suggest that the progenitors that are born in the human SVZ migrate via the rostral migratory stream (RMS) towards the olfactory bulb (OB), similar to what has been observed in other mammals. A subpopulation of astrocytes in the SVZ specifically expresses an assembly‐compromised isoform of the intermediate filament protein glial fibrillary acidic protein (GFAP‐δ). To further define the phenotype of these GFAP‐δ expressing cells and to determine whether these cells are present throughout the human subventricular neurogenic system, we analysed SVZ, RMS and OB sections of 14 aged brain donors (ages 74‐93). GFAP‐δ was expressed in the SVZ along the ventricle, in the RMS and in the OB. The GFAP‐δ cells in the SVZ co‐expressed the neural stem cell (NSC) marker nestin and the cell proliferation markers proliferating cell nuclear antigen (PCNA) and Mcm2. Furthermore, BrdU retention was found in GFAP‐δ positive cells in the SVZ. In the RMS, GFAP‐δ was expressed in the glial net surrounding the neuroblasts. In the OB, GFAP‐δ positive cells co‐expressed PCNA. We also showed that GFAP‐δ cells are present in neurosphere cultures that were derived from SVZ precursors, isolated postmortem from four brain donors (ages 63‐91). Taken together, our findings show that GFAP‐δ is expressed in an astrocytic subpopulation in the SVZ, the RMS and the OB. Importantly, we provide the first evidence that GFAP‐δ is specifically expressed in longterm quiescent cells in the human SVZ, which are reminiscent of NSCs.     

Histone deacetylase inhibitors de-repress tyrosine hydroxylase expression in the olfactory bulb and rostral migratory stream

Most olfactory bulb (OB) interneurons are derived from neural stem cells in the subventricular zone (SVZ) and migrate to the OB via the rostral migratory stream (RMS). Mature dopaminergic interneurons in the OB glomerular layer are readily identified by their synaptic activity-dependent expression of tyrosine hydroxylase (TH). Paradoxically, TH is not expressed in neural progenitors migrating in the RMS, even though ambient GABA and glutamate depolarize these progenitors. In forebrain slice cultures prepared from transgenic mice containing a GFP reporter gene under the control of the Th 9 kb upstream regulatory region, treatment with histone deacetylase (HDAC) inhibitors (either sodium butyrate, Trichostatin A or Scriptaid) induced Th-GFP expression specifically in the RMS independently of depolarizing conditions in the culture media. Th-GFP expression in the glomerular layer was also increased in slices treated with Trichostatin A, but this increased expression was dependent on depolarizing concentrations of KCl in the culture media. Th-GFP expression was also induced in the RMS in vivo by intra-peritoneal injections with either sodium butyrate or valproic acid. Quantitative RT-PCR analysis of neurosphere cultures confirmed that HDAC inhibitors de-repressed Th expression in SVZ-derived neural progenitors. Together, these findings suggest that HDAC function is critical for regulating Th expression levels in both neural progenitors and mature OB dopaminergic neurons. However, the differential responses to the combinatorial exposure of HDAC inhibitors and depolarizing culture conditions indicate that Th expression in mature OB neurons and neural progenitors in the RMS are regulated by distinct HDAC-mediated mechanisms.     

Drebrin Regulates Neuroblast Migration in the Postnatal Mammalian Brain

After birth, stem cells in the subventricular zone (SVZ) generate neuroblasts that migrate along the rostral migratory stream (RMS) to become interneurons in the olfactory bulb (OB). This migration is crucial for the proper integration of newborn neurons in a pre-existing synaptic network and is believed to play a key role in infant human brain development. Many regulators of neuroblast migration have been identified; however, still very little is known about the intracellular molecular mechanisms controlling this process. Here, we have investigated the function of drebrin, an actin-binding protein highly expressed in the RMS of the postnatal mammalian brain. Neuroblast migration was monitored both in culture and in brain slices obtained from electroporated mice by time-lapse spinning disk confocal microscopy. Depletion of drebrin using distinct RNAi approaches in early postnatal mice affects neuroblast morphology and impairs neuroblast migration and orientation in vitro and in vivo. Overexpression of drebrin also impairs migration along the RMS and affects the distribution of neuroblasts at their final destination, the OB. Drebrin phosphorylation on Ser142 by Cyclin-dependent kinase 5 (Cdk5) has been recently shown to regulate F-actin-microtubule coupling in neuronal growth cones. We also investigated the functional significance of this phosphorylation in RMS neuroblasts using in vivo postnatal electroporation of phosphomimetic (S142D) or non-phosphorylatable (S142A) drebrin in the SVZ of mouse pups. Preventing or mimicking phosphorylation of S142 in vivo caused similar effects on neuroblast dynamics, leading to aberrant neuroblast branching. We conclude that drebrin is necessary for efficient migration of SVZ-derived neuroblasts and propose that regulated phosphorylation of drebrin on S142 maintains leading process stability for polarized migration along the RMS, thus ensuring proper neurogenesis.     

Neurogenesis and neuronal migration in the neonatal rat forebrain anterior subventricular zone do not require GFAP-positive astrocytes

A prolific neuronal progenitor cell population in the anterior portion of the neonatal rat forebrain subventricular zone, the SVZa, is specialized for the production of olfactory bulb interneurons. At all ages, SVZa-derived cells traverse a tangential migratory pathway, the rostral migratory stream (RMS), while en route to the olfactory bulb. Unlike other neuronal progenitor cells of the forebrain, migrating progeny of SVZa progenitors express neuronal-specific proteins and continue to divide into adulthood. Recent studies indicate that in the adult, migrating SVZa-derived cells are ensheathed by astrocytes, although the function of these astrocytes has not been determined. To explore the possible role(s) of astrocytes in the rat SVZa and RMS, we examined the expression of astroglial-specific genes in the postnatal SVZa and RMS using RT-PCR, in situ hybridization, and immunohistochemistry during (Postnatal Days 1-10) and after the period of peak olfactory bulb interneuron generation. We also examined the expression of neuronal-specific genes throughout the rostral-caudal extent of the postnatal subventricular zone to determine if differential cell type-specific gene expression could distinguish the neurogenic SVZa as a region distinct from the remainder of the SVZ. We found little to no astrocyte-specific gene expression in the P0-P7 SVZa, although the neuron-specific isoforms of tubulin (T alpha 1 and beta-III tubulin) were expressed abundantly in the SVZa and RMS. In contrast, astrocyte-specific genes were strongly expressed in the SVZ posterior to the SVZa. GFAP expressions begins to appear in some restricted areas of the rostral migratory stream after the first postnatal week. These data suggest that astroglia are not involved in the generation or migration of most olfactory bulb interneurons. Moreover, the scarcity of glial markers in the neonatal SVZa indicates that the forebrain subventricular zone includes a distinct neurogenic anterior region containing predominantly committed neuronal progenitor cells.     

Adult-generated neurons exhibit diverse developmental fates

In mammals, olfactory bulb (OB) interneurons, granule cells and periglomerular (PG) cells, are generated throughout adulthood. PG cells comprise a heterogeneous population in both morphology and molecular phenotypes. It is unknown whether adult genesis of PG cells occurs among all subtypes or is limited to a subpopulation. We show that within 2 weeks after retroviral labeling in the subventricular zone, two morphological populations of PG cells are found in the OB, one with large multi-glomerular dendritic arbors, and one with dendritic arbors limited to one or two glomeruli. On both types, immature dendritic spines are first evident at 4 weeks and mature, pedunculated spines by 6 weeks. To differentiate PG subpopulations we used expression of calcium binding proteins, GAD67 and tyrosine hydroxylase as markers. Among adult-born BrdU labeled cells, all molecular subtypes were represented, although GAD67 and tyrosine hydroxylase expressing cells were overrepresented proportional to their expression in the total PG cell population. During the time when spines are maturing, approximately half the PG cells are lost, in roughly equal proportions to their generation. Our data show the diverse developmental potential of SVZ neuroblasts and suggest that integration into synaptic circuits is necessary for survival.     

Changes in cell migration and survival in the olfactory bulb of the pcd/pcd mouse

Postnatally, the Purkinje cell degeneration mutant mice lose the main projecting neurons of the main olfactory bulb (OB): mitral cells (MC). In adult animals, progenitor cells from the rostral migratory stream (RMS) differentiate into bulbar interneurons that modulate MC activity. In the present work, we studied changes in proliferation, tangential migration, radial migration patterns, and the survival of these newly generated neurons in this neurodegeneration animal model. The animals were injected with bromodeoxyuridine 2 weeks or 2 months before killing in order to label neuroblast incorporation into the OB and to analyze the survival of these cells after differentiation, respectively. Both the organization and cellular composition of the RMS and the differentiation of the newly generated neurons in the OB were studied using specific markers of glial cells, neuroblasts, and mature neurons. No changes were observed in the cell proliferation rate nor in their tangential migration through the RMS, indicating that migrating neuroblasts are only weakly responsive to the alteration in their target region, the OB. However, the absence of MC does elicit differences in the final destination of the newly generated interneurons. Moreover, the loss of MC also produces changes in the survival of the newly generated interneurons, in accordance with the dramatic decrease in the number of synaptic targets available.     

A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons

GABAergic inhibitory neurons are a large population of neurons in the central nervous system (CNS) of mammals and crucially contribute to the function of the circuitry of the brain. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. While a number of mouse lines expressing the green fluorescent protein (GFP) in different subpopulations of GABAergic cells are available, GFP expressing mouse lines are not suitable for either crossbreeding to other mouse lines expressing GFP in other cell types or for Ca²⁺-imaging using the superior green Ca²⁺-indicator dyes. Therefore, we have generated a novel transgenic mouse line expressing the red fluorescent protein tdTomato in GABAergic neurons using a bacterial artificial chromosome based strategy and inserting the tdTomato open reading frame at the start codon within exon 1 of the GAD2 gene encoding glutamic acid decarboxylase 65 (GAD65). TdTomato expression was observed in all expected brain regions; however, the fluorescence intensity was highest in the olfactory bulb and the striatum. Robust expression was also observed in cortical and hippocampal neurons, Purkinje cells in the cerebellum, amacrine cells in the retina as well as in cells migrating along the rostral migratory stream. In cortex, hippocampus, olfactory bulb and brainstem, 80% to 90% of neurons expressing endogenous GAD65 also expressed the fluorescent protein. Moreover, almost all tdTomato-expressing cells coexpressed GAD65, indicating that indeed only GABAergic neurons are labelled by tdTomato expression. This mouse line with its unique spectral properties for labelling GABAergic neurons will therefore be a valuable new tool for research addressing this fascinating cell type.     

GABA and glutamate signaling: homeostatic control of adult forebrain neurogenesis

The neurotransmitter GABA exerts a strong negative influence on the production of adult-born olfactory bulb interneurons via tightly regulated, non-synaptic GABAergic signaling. After discussing some findings on GABAergic signaling in the neurogenic subventricular zone (SVZ), we provide data suggesting ambient GABA clearance via two GABA transporter subtypes and further support for a non-vesicular mechanism of GABA release from neuroblasts. While GABA works in cooperation with the neurotransmitter glutamate during embryonic cortical development, the role of glutamate in adult forebrain neurogenesis remains obscure. Only one of the eight metabotropic glutamate receptors (mGluRs), mGluR5, has been reported to tonically increase the number of proliferative SVZ cells in vivo, suggesting a local source of glutamate in the SVZ. We show here that glutamate antibodies strongly label subventricular zone (SVZ) astrocytes, some of which are stem cells. We also show that some SVZ neuroblasts express one of the ionotropic glutamate receptors, AMPA/kainate receptors, earlier than previously thought. Collectively, these findings suggest that neuroblast-to-astrocyte GABAergic signaling may cooperate with astrocyte-to-neuroblast glutamatergic signaling to provide strong homeostatic control on the production of adult-born olfactory bulb interneurons. An erratum to this article can be found at     

Identification and characterization of neuroblasts in the subventricular zone and rostral migratory stream of the adult human brain

It is of great interest to identify new neurons in the adult human brain, but the persistence of neurogenesis in the subventricular zone (SVZ) and the existence of the rostral migratory stream (RMS)-like pathway in the adult human forebrain remain highly controversial. In the present study, we have described the general configuration of the RMS in adult monkey, fetal human and adult human brains. We provide evidence that neuroblasts exist continuously in the anterior ventral SVZ and RMS of the adult human brain. The neuroblasts appear singly or in pairs without forming chains; they exhibit migratory morphologies and co-express the immature neuronal markers doublecortin, polysialylated neural cell adhesion molecule and βIII-tubulin. Few of these neuroblasts appear to be actively proliferating in the anterior ventral SVZ but none in the RMS, indicating that neuroblasts distributed along the RMS are most likely derived from the ventral SVZ. Interestingly, no neuroblasts are found in the adult human olfactory bulb. Taken together, our data suggest that the SVZ maintains the ability to produce neuroblasts in the adult human brain.     

New GABAergic interneurons in the adult neocortex and striatum are generated from different precursors

Ongoing neurogenesis in the adult mammalian dentate gyrus and olfactory bulb is generally accepted, but its existence in other adult brain regions is highly controversial. We labeled newly born cells in adult rats with the S-phase marker bromodeoxyuridine (BrdU) and used neuronal markers to characterize new cells at different time points after cell division. In the neocortex and striatum, we found BrdU-labeled cells that expressed each of the eight neuronal markers. Their size as well as staining for gamma-aminobutyric acid (GABA), glutamic acid decarboxylase 67, calretinin and/or calbindin, suggest that new neurons in both regions are GABAergic interneurons. BrdU and doublecortin-immunoreactive (BrdU+/DCX+) cells were seen within the striatum, suggesting migration of immature neurons from the subventricular zone. Surprisingly, no DCX+ cells were found within the neocortex. NG2 immunoreactivity in some new neocortical neurons suggested that they may instead be generated from the NG2+ precursors that reside within the cortex itself.     

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.     

The spatiotemporal segregation of GAD forms defines distinct GABA signaling functions in the developing mouse olfactory system and provides novel insights into the origin and migration of GnRH neurons

Gamma‐aminobutyric acid (GABA) has a dual role as an inhibitory neurotransmitter in the adult central nervous system (CNS) and as a signaling molecule exerting largely excitatory actions during development. The rate‐limiting step of GABA synthesis is catalyzed by two glutamic acid decarboxylase isoforms GAD65 and GAD67 coexpressed in the GABAergic neurons of the CNS. Here we report that the two GADs show virtually nonoverlapping expression patterns consistent with distinct roles in the developing peripheral olfactory system. GAD65 is expressed exclusively in undifferentiated neuronal progenitors confined to the proliferative zones of the sensory vomeronasal and olfactory epithelia In contrast GAD67 is expressed in a subregion of the nonsensory epithelium/vomeronasal organ epithelium containing the putative Gonadotropin‐releasing hormone (GnRH) progenitors and GnRH neurons migrating from this region through the frontonasal mesenchyme into the basal forebrain. Only GAD67+, but not GAD65+ cells accumulate detectable GABA. We further demonstrate that GAD67 and its embryonic splice variant embryonic GAD (EGAD) concomitant with GnRH are dynamically regulated during GnRH neuronal migration in vivo and in two immortalized cell lines representing migratory (GN11) and postmigratory (GT1–7) stage GnRH neurons, respectively. Analysis of GAD65/67 single and double knock‐out embryos revealed that the two GADs play complementary (inhibitory) roles in GnRH migration ultimately modulating the speed and/or direction of GnRH migration. Our results also suggest that GAD65 and GAD67/EGAD characterized by distinct subcellular localization and kinetics have disparate functions during olfactory system development mediating proliferative and migratory responses putatively through specific subcellular GABA pools. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 249–270, 2015