1.1 Overview

During embryonic development, neural stem cells (NSCs) are the subset of stem cells that give rise to the entire framework and cell types in the central nervous system. By definition, they are a set of multipotent cells that are capable of self-proliferation and of differentiating into new neurons and glial cell. In the past decades, adult neurogenesis, a process that refers to the development of new neurons and glia from a restrict division of adult neural precursors was discovered, that shone light on the study of post-embryonic neural development and potential clinical applications.



1.2 NSCs in Adult Mammalian brains

In adult mammalian brains, the regions that neurogenesis occurs were found to be restricted to two sites: the subgranular zone (SGZ) of the dentate gyrus in the hippocampus that generates new granule cells; and the subventricular zone (SVZ) in lateral ventricle where newly differentiated neurons migrate through rostral migratory sytem (RMS) to the olfactory bulb [1,2,3]. These residential adult NSCs proliferate to replenish and also differentiate into neurons, which are usually referred to as adult-born neurons [4,5,6].
ANSCs.jpg
Fig 1. Anatomical view of adult NSCs niches in rodent brain

1.2.1 SGZ in Hippocampal Dentate Gyrus

Adult hippocampal neurogenesis originates from the population of adult NSCs in the sub granular zones (SGZ) of the dentate gyrus. In the SGZ, it appears that multiple types of glial-like cells coexisted as residential adult NSCs, based on their abilities to differentiation, stem-cell specific cellular markers and receptors. According to the marker expressions, the adult NSCs can be divided into two groups:
  • a) Type 1 cells, also called radial glia-like cells, are characterized by the expression of nestin (Nes), glial fibrillary acidic protein (GFAP), brain lipid-binding protein (Blbp), glutamate transporter (Glast), and Sox2. [7]; Recent studies using inducible Cre-recombinase driven by various promoter including Glia GFAP, Nestin, and GLAST have provided evidence substantiating that type 1 cells are the primary NSCs in the adult brain [8].
  • b) Type 2 cells, also called nonradial precursor cells, display a more horizontal morphology with very short processes. It is believed that the nonradial cells, derived from radial glia-like cells, are highly proliferative. The nonradial cells are characterized by their expression of Nes and Sox2 [7]. Type 2 cells can be further divided into groups based on their pro-neuronal transcription factor expression: i). Type 2a cells that express Mash1 and ii) Type 2b cells expressing Prox1 andNeurod1 [9]. Type 2b cells are the transient amplifying progenitors(TAPs) in the SGZ, the intermediate cell type between NSC and neuroblasts.

SGZ.jpg

Fig 2. Schematic of neurogenesis processes in Adult hippocampus.[2]. The process starts with type 1 cell/radial glia-like cell, to progenitors and then neuroblast. The neuroblask migrate out of the SGZ and differentiate into neurons in the pre-exisiting neuronal network in the hippocampus/dentate gyrus.


1.2.2 SVZ in the Lateral Ventricle

In the SVZ in the lateral ventricle, two types of glia-like cells have been proposed to be the local adult NSCs.
  • a) Type B cells line along the ependymal cells on the lateral ventricle, between the ventricular membrane and migrating neuroblasts. They are the quiescent NSCs that are Nes negative and GFAP positive. Type B stem cells can be further divided in two groups: type B1 and type B2 cells. Type B1 and B2 cells serves the roles to physically separate the neuroblast (type A cells) from the ependymal layer and surrounding striatal parenchyma, respectively. A recent lineage genetics study showed that type B1 cells belong to the category of quiescent NSCs [10], whereas type B2 cells were more proliferative.
  • b) Type C cells, differentiated from type B cells, are the most actively proliferating cells. They are also the TAPs in the SVZ region, which give rise to neuroblast (type A cells).

SVZ2.jpg

Fig 3. Schematic of neurogenesis processes in Adult SVZ in lateral ventricle.[2]. The process starts with type B cell/radial glia-like cell, to TAPs and then neuroblast. The neuroblask migrate through RMS and differentiate into neurons in the pre-exisiting neuronal network in the olfactory bulb.

1.2.3 Controversies on Cell Classifications
A recent study using the canonical Notch signaling reporter, Hes5, to label adult NSCs in the SGZ revealed the presence of two groups depending on theor activity: (a) quiescent NSCs that retains BrdU pulse; and 2) proliferating horizontal type I cells, the active adult NSCs that express proliferating cell nuclear antigen (Pcna) exhibit BrdU pulse [9]. This sorting method disregarded the morphological and molecular characterization, but rather focused on the proliferation profile.

After a decade of controversy, a type of enpendymal cells found in the SVZ that are CD133/prominin1 positive were demonstrated to be multipotent [11]. It was demonstrated that ischemia can cause the activation of this type of ependymal cells to generate neuroblasts and astrocytes, providing evidence of these cells to be adult NSCs [12 Carlen 2009]. Recently, a study using the split Cre-recombinase also characterized a type of radial glial-like cells in the SVZ that are CD133/prominin1 and GFAP double positive, which extend processes basally between ependymal cells and ventricle, apically through TAPs and neuroblasts, were also quiescent NSCs [12]. It is still under debate whether the previously identified CD133/prominin1 positive ependymal stem cells from experiment artifacts such as comtamination of CD133 and GFAP double positive cells. It is possible that the identity of SVZ quiescent NSCs can have a much broader criteria [13].



1.3 Adult Neurogenesis Pathways

In general, adult neurogenesis involves the differentiating progress from quiescent adult NSCs to transient amplifying progenitors (TAPs) and finally to neuroblasts. The neuroblasts then migrate to other regions of the brain, and give rise to adult-born neurons that can integrate into existing neural circuitry.

1.3.1 Adult Neurogenesis in Hippocampus

In the adult SGZ, both type 1 and type 2 cells (radial and nonradial glia-like cells) can give rise to intermediate TAPs, which in turn generate neuroblasts (Figure 2). Immature neurons then migrate into the inner granule cell layer and differentiate into dentate granule cells in the hippocampus, followed by dendrites extensions and axonal projections towards CA3 regions [14]. New neurons eventually incorporate into existing neuronal circuitry through a stereotypic synaptic integration [15]. Initially, these adult-born neurons received tonic activation by ambient GABA released from surrounding interneurons [16,17]. Later on, they started to receive GABAergic synaptic inputs, followed by glutamatergic inputs ([17] [18]Overstreet-Wadiche et al., 2006b) and mossy fiber synaptic outputs to CA3 neurons [19]. During specific differentiation stages, adult-born neurons were found to display higher excitability and ehanced synaptic plasticity comparing to granule cells that already exhisted in the circuitry [15]. After maturation has taken place, adult-born neurons exhibit similar electrophysiological properties as mature neurons, specifically firing behaviour upon receiving GABAergic and glutamatergic inputs [20]. However, whether molecular or chemical behaviour differs between neurons born at different developmental stage remains unknown.

1.3.2 Adult Neurogenesis in Olfactory Bulb


In the adult SVZ, self-proliferating radial glia-like cells give rise to transient amplifying cells, which later differentiate into neuroblasts (Figure 2). The neuroblasts migrate through the rostral migratory stream (RMS) to the olfactory bulb [21]. Upon approaching the core structure of olfactory bulb, the pre-neurons blasts detach from the RMS and migrate toward glomeruli where they then differentiate into different subtypes of interneurons [2]. The majority of these adult-born neurons become GABAergic granule neurons to form dendro-dendritic synapses with mitral cells. A minority become GABAergic periglomerular neurons, a small percentage of which also receive dopaminergic input. One study suggests that a very small percentage of new neurons develop into glutamatergic juxtaglomerular neurons [21] Br. Analysis of labeled precursors and newborn neurons by electrophysiology and confocal imaging, including live imaging in vivo, have revealed physiological properties and sequential stages of neuronal development and synaptic integration (Figure 3) [2].

1.3.3 Adult NSCs Activation

External stimuli were found to activate the quiescent aNSCs. For example, exercising (e.g. running) recruits quiescent NSCs into the active NSC pool [13]. Depletion of active NSCs rather than quiescent NSCs leads to impaired neurogenesis in aged mice. Furthermore, if stimulated by seizure, the quiescent NSCs in aged mice can be reactivated [9].
Furthermore, some studies have demonstrated that the quiescence of SGZ adult NSCs were maintained by BMP signaling. This brought up the question that whether Wnt signaling pathway which is usually important in proliferation of cells, is involved in the regulation of adult NSCs. Previous studies reported that Wnt signaling was activated in the SGZ progenitors and in coordination with Sox2 promoted the proliferation and neuronal differentiation of hippocampal progenitors through inducing the expression of Neurod1 [21]. These results indicate that Wnts may act as a key signal to activate NSCs in the hippocampus.


1.4 Functions of Adult Neurogenesis

Most studies so far provided evidence that the newly generated neurons support learning and memory at hippocampus level and olfaction, corresponding to the two niches where neural precursors reside. However, it still remains controversial whether adult neurogenesis occurs in other region of brain, such as neocortex. At the same time, other functionalities of these neural stem cells such as injury recover and roles in neurodegenerative diseases are not sufficiently clear but under intensive study. The activation of neurogenesis process is attributed by numerous intrinsic and environmental pathways, revealing the complexity and high level of regulations in adult brain.

1.4.1 Roles in hippocampal-dependent activities

Current evidence substantiates that rather than merely replace the neurons loss in the hippocampus in adult mammalian brain, the adult-born neurons are continuously added into the existing neuronal circuitry as it responds to various experiences throughout life (15). Adult-born neurons exhibit unique transient properties that are distinct from mature neurons development.

1.4.1.1 Hippocampal Synaptic Plasticity and Circuitry Modulation

First of all, newly development neurons resulted from adult neurogenesis displayed different excitability in response to action potential and neurotransmitters [21]. For instance, a group has shown that adult NSCs display resistance to high-input and upregulation of low threshold Calcium channels which leads to elavated excitability and firing rate following weak excitatory inputs. Secondly, associative long-term potentiation (LTP) can be more easily induced in newly developed neurons than in mature neurons under identical conditions, indicating a potentially enhanced synaptic plasticity. However, this increased LTP inducibility, results from both elevated LTP amplitude and decreased LTP induction threshold, is present only within a narrow time frame: namely between 4-6 weeks of cell age [21]. Overall, this unique and transient property allows adult-born neurons to function differently in parallel or in sequence with existing neurons, therefore bringing about new characteristics in the microcircuits, adding a whole new level of plasticity. Since they are added into an built neuronal framework, adult-born neurons may be an important player in modulate activity at the circuitry level as discribed in Fig 4.

Cir.jpg
  • Fig 4. Demonostration of pattern seperation [Figure from 21]. Green: newly integrated neurons.]

1.4.1.2 Effect on Learning and Memory

In conjunction with the effect of adult-born neurons in hippocampal circuitry, subsequent studies have demonstrated the importance of hippocampal neurogenesis during adulthood on learning and memory. The loss-of-functions approach were often utilized in early studies, two major techniques included 1) anti-mitotic drugs, such as methylazoxymethonol (MAM) or 2). X-rays. Several noteworthy pioneers in the field provided several indications in potential deficits that can arise due to the lack of adult neurogenesis:
  1. MAM-treated mice displayed severe deficits in some (trace eye-blink conditioning and trace fear conditioning), but not all hippocampal-dependent tasks (contextual fear conditioning) [22].
  2. MAM treated mice also lost long-term memory reinforcement normally induced by environmental cues [23].
  3. Mice treated with cranial radiation displayed severe deficits in many hippocampal-dependent tasks including: T-maze place recognition, spatial learning in Barnes maze, contextual fear conditioning, and nonmatching-to-sample (NMTS) tasks, whereas water maze spatial learning, and hippocampus- independent tasks were largely unaffected (Reviewed in 8)

However, these approaches came with inherited short-comings: the inhibition of mitosis, or the loss of neurons, are not anatomically restricted to the hippocampus due to the nature of the interventions, therefore, nonspecific neuronal loss may attribute in part to the behavioral deficits observed. At current stage, most studies make use of genetic interventions to interfere with adult neurogenesis in animal models. Sahay's group has conducted several significant experiments in the field, that conjointly suggested that adult neurogenesis at hippocampal level is crucial in spatial pattern separation, contextual fear conditioning, clearance of hippocampal memory traces, and reorganization of memory ([21], reviewed in [24]). Collectively with more recent computational approaches, there are increasing amount of evidence to substantiate the role of adult hippocampal neurogenesis in fine-tuning learning and memory.

1.4.1.3 Mood Regulation

Adult neurogenesis has also been suggested to attribute to higher brain function, such as mood regulation. Santarelli et al. (2003) were the first to demonstrated that the loss of SGZ neurogenesis quenched the effectiveness of antidepressants at a behavioral level (25). However, there still exists great variability on the depression animal model, on top of the complex causal-effect from neurogenesis on behavior. Therefore, it is still controversial whether depletion of hippocampal neurogenesis is a pathological factor for depression at this stage.

1.4.2 Roles in odorant-dependent learning

The regulation of olfaction-related learning and memory by adult-born neurons is exerted at three levels:
  1. Odor acuity is in part regulated by pattern separation processes in the olfactory bulb which is dependent on inhibitory circuits, many of which are fine-tuned through adult neurogenesis.[Adult-born neurons from SVZ usually integrate into the olfactory bulb as two types of cells, the juxtaglomerular neurons and inhibitory granule cells Collectively they play a part in lateral inhibition and the spatiotemporal structure of olfactory bulb output [21]. This inhibition helps enhance contrast and ultimately, the separation between similar patterns of olfactory input [25].
  2. It was suggested that the survival of adult born gruanule cells are tightly associated with odorant-related memory and enhanced olfactory acuity. Reduced or disrupted olfactory bulb neurogenesis disturbs normal synaptic inhibition also impairs odor-evoked activity. ([26] Moreno et al 2009)
  3. In hippocampus, as demonstrated in previous section, newly-born granule cells display higher synaptic plasticity than mature granule cells. Likewise, in OB, young granule cells exhibit more robust synaptic plasticity and are more responsive to novel odors. Song et al (2011) reviewed that since adult-born neurons synapses onto most cell types in the olfactory bulb, this network of neurons may be specifically important at manipulate reponses to novel odors. However, most manipulations of adult neurogenesis did not necessarily cause impairments in olfactory discrimination. Only few studies, the role for adult-born granule cells was suggested in olfactory discrimination [26]. It is possible that adult-born granule cells in serves as a rather elegant manipulation that is only detectable only in the more sophisticated behavior tests. It seems like more studies and new approaches are yet to be done in order to obtain qualitative information in order to pin down the function of adult-born neurons in olfactory system.

1.4.3 Potential roles in CNS injury repair

Between quiescent adult NSCs and TAPS, evidence has substantiated that an “active” form of NSCs may exist function as an intermediate.
Like the quiescent NSCs, active NSCs are multipotent and self-renewing but highly proliferative. The advantage of this ‘‘coexistence’’ is that quiescent NSCs act as a reservoir of adult NSCs while active NSCs can be utilized for CNS homeostasis and regeneration.
A study has described that upon injury, active adult NSCs pool can be upregulated quickly to restore damaged tissue and downregulated after regeneration processes. The response of Wnt reactivated progenitors has been shown to be essential for retina regeneration [25]. Therefore, the characterization of active NSCs and molecular mechanism, which can shift quiescence adult NSCs into their active form, may provide therapeutic benefits for realizing adulthood CNS regeneration.




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