Synaptogenesis in the Adult CNS – Hippocampus

C H A P T E R

38 Synaptogenesis in the Adult CNS – Hippocampus C. Zhao1*, N. Toni2*, F.H. Gage1 1

Salk Institute for Biological Studies, La Jolla, CA, USA; 2University of Lausanne, Lausanne, Switzerland; * Contributed equally

O U T L I N E 38.1 Overview of the Hippocampus and Adult Neurogenesis in the Dentate Gyrus 38.1.1 A Brief Introduction to the Hippocampus 38.1.2 The Functional Significance of Hippocampal Neurogenesis 38.1.3 Methods for Studying the Integration of Newborn Granule Cells in the Adult Hippocampus 38.1.3.1 BrdU and Molecular Markers 38.1.3.2 Retroviral Vectors 38.2 Synaptogenesis in the Adult Hippocampus – Newborn Granule Cells 38.2.1 An Overview of the Maturation Process of Newborn Granule Cells 38.2.2 Synaptogenesis on Dendritic Spines of Newborn Granule Cells 38.2.3 Synaptogenesis of Axons from Newborn Granule Cells 38.2.4 Regulation of Synaptogenesis by Experience and Disease 38.2.4.1 Exercise 38.2.4.2 Learning 38.2.4.3 Antidepression Treatments 38.2.4.4 Epilepsy

Cellular Migration and Formation of Neuronal Connections: Comprehensive Developmental Neuroscience, Volume 2 http://dx.doi.org/10.1016/B978-0-12-397266-8.00111-3

38.2.4.5 Aging and Neurodegenerative Diseases

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38.2.5 Signaling Molecules that Regulate Synaptogenesis of Newborn Granule Cells 38.2.5.1 GABA 38.2.5.2 Glutamate 38.2.5.3 Other Neurotransmitters 38.2.5.4 Other Molecules that Regulate Synapse Formation of Newborn Granule Cells 38.2.6 Theoretical and Computational Models of Synaptogenesis of Newborn Cells and Their Function in the Adult Hippocampal Neurogenesis

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38.3 Synaptogenesis in the Adult Hippocampus – CA1 and CA3 Pyramidal Cells and Other Neurons of the Hippocampus

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38.4 Conclusions

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# 2013 Elsevier Inc. All rights reserved.

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38.1 OVERVIEW OF THE HIPPOCAMPUS AND ADULT NEUROGENESIS IN THE DENTATE GYRUS 38.1.1 A Brief Introduction to the Hippocampus The hippocampus is best known for its role in learning and memory. It is a major part of the limbic system and is located inside the medial temporal lobe. The hippocampus mainly consists of three anatomical parts: CA1, CA3, and the dentate gyrus (DG). The DG receives its major excitatory input from the entorhinal cortex and then projects to the CA3, whose pyramidal neurons project to the CA1. The pyramidal cells in the CA1 then send the major output of the hippocampus back to the entorhinal cortex through the subiculum. Most of the neurons in the hippocampus are generated during early development, with the exception of the granule cells in the DG, which are generated at peak level during early postnatal days and are continuously added to the granule cell layer (GCL) at a low level throughout the life of mammals, including humans (Altman and Das, 1965; Eriksson et al., 1998) (Figure 38.1). The other brain area where adult

neurogenesis occurs unambiguously is the subventricular zone, from which adult-born neurons migrate to the olfactory bulb and participate in olfaction. Neurogenesis in the adult DG represents a unique form of plasticity in the hippocampus, which most likely contributes to the specific function of the DG. There are two different kinds of synaptic plasticity in the adult hippocampus. One is similar to other brain areas and involves the formation/elimination and strengthening/weakening of synapses on existing neurons; it will be briefly described at the end of this chapter. The second type of synaptic plasticity, which is the focal point of this chapter, involves the de novo formation and modification of synapses on newly added granule cells in the DG.

38.1.2 The Functional Significance of Hippocampal Neurogenesis The level of neurogenesis in the adult DG is readily influenced by daily activities and physiological and pathological conditions, such as diet, exercise, stress, aging, and neurodegenerative diseases. In general, the level of

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FIGURE 38.1 Adult-born granule neurons integrate in the dentate gyrus (DG). This confocal micrograph of a horizontal section of the mouse hippocampus shows adult-born neurons labeled with retroviral-induced GFP expression (green) in the DG. Astrocytes are immunolabeled in red and cell nuclei in blue. The dendrites of newborn neurons extend in the molecular layer (ml) where they receive axo-spinous excitatory input from the entorhinal cortex, a three-dimensional reconstruction of which is shown in the insert. In the insert, a dendritic spine of an adult-born neuron, shown in green, contacts a bouton from a perforant path axon (blue), which also contacts a dendritic spine from another granule neuron (red), thereby forming a multiple-synapse bouton. Yellow: presynaptic vesicles. The axons from the adult-born neurons extend into the hilus (hl) and the CA3, where they contact dendrites of pyramidal neurons. The insert in the CA3 shows a three-dimensional reconstruction of a mossy terminal (green) contacting a thorny excrescence from a CA3 pyramidal neuron (red), itself contacting a mossy terminal from another neuron (blue).

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38.1 OVERVIEW OF THE HIPPOCAMPUS AND ADULT NEUROGENESIS IN THE DENTATE GYRUS

hippocampal neurogenesis correlates with an animal’s performance of behavioral tasks that are designed to test hippocampus-dependent learning and memory. To examine the functional role of adult hippocampal neurogenesis directly, several methods were developed to target hippocampal neurogenesis, including X-ray irradiation, antimitotic agents, and genetic mouse models (Deng et al., 2009; Shors et al., 2001; Snyder et al., 2005, reviewed in Deng et al., 2010). Although discrepancies exist, these studies suggest that adult hippocampal neurogenesis contributes to certain forms of hippocampusdependent learning and memory and may also participate in mood regulation (reviewed in Deng et al., 2010; Zhao et al., 2008). Because neurogenesis specifically occurs in the DG, but in no other area of the hippocampus, recent experimental studies and computational modeling have focused on the putative contribution of neurogenesis to pattern separation, a specific function attributed to the DG (Clelland et al., 2009, reviewed in Aimone et al., 2010).

38.1.3 Methods for Studying the Integration of Newborn Granule Cells in the Adult Hippocampus Soon after the discovery of adult neurogenesis, it became clear that adult-born neurons receive synaptic input. Using tritiated thymidine and electron microscopy, axosomatic synapses were observed on adult-born neurons in rats 30 days after cell birth (Kaplan and Hinds, 1977). However, the limitations of the technique used at that time did not allow for analysis of the whole cell morphology. It is only now, a few decades later, following the immunohistochemical detection of a nucleic acid analogue, 5-bromo-2-deoxyuridine (BrdU), and the design of genetic-based techniques that enable the visualization of entire adult-born neurons, that analysis of the development and connectivity of synapses, both at the morphological and electrophysiological levels, became possible. 38.1.3.1 BrdU and Molecular Markers BrdU is a thymidine analogue that is incorporated into newly synthesized DNA. It can be administered systemically, either per os or by intraperitoneal injections, has a half-life of a few hours, and can be detected immunohistochemically. As such, this method is noninvasive and an efficient means of labeling a population of newly generated cells. It has been extensively used to analyze cell proliferation and survival (Kuhn et al., 1996; Taupin, 2007). Because of its localization into the nucleus, BrdU labeling is not suitable for morphological analyses. However, by combining BrdU and immature neuronal markers that are expressed in dendritic and axonal processes of immature neurons (e.g., doublecortin

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and polysialylated-neural cell adhesion molecule (PSA-NCAM)), one can determine the early morphological features associated with BrdU-labeled cells using immunohistochemistry at both light- and electronmicroscopic levels. These markers also have been used after electrophysiological analyses to determine the identity of the recorded cells (Schmidt-Hieber et al., 2004). Recently, the BrdU approach also has been combined with markers of neuronal activity (such as c-fos, Zif268, and Arc) to analyze the recruitment of newborn neurons (Kee et al., 2007; Tashiro et al., 2007; Trouche et al., 2009). Although this approach does not directly examine synapse formation, the activation of new neurons under these conditions suggests the integration of these cells into a specific network. 38.1.3.2 Retroviral Vectors Although BrdU is a good method to date the birth of and label newborn cells, its requirement of immunohistochemical treatment hinders the visualization of living cells. Furthermore, immature neuronal markers are visible only during a specific time window (4 weeks and younger). Retrovirus labeling overcomes these caveats, although the delivery of virus is rather invasive and is not as efficient. Most of the commonly used retrovirus vectors in the field of adult neurogenesis are derived from the Moloney murine leukemia virus (MoMLV), which belongs to a type of RNA virus that has reverse transcriptase activity that converts the RNA virus genome into double-stranded DNA, known as the provirus, in host cells. The DNA provirus of MoMLV does not enter the cell nucleus on its own; rather, it requires breakdown of the nuclear membrane during mitosis to transduce the infected cell in a stable manner, allowing the specific labeling of proliferating cells that enter mitosis shortly after virus delivery. These viruses have been engineered so that they do not replicate in the host cells and express ectopic genes such as the gene-encoding green fluorescent protein (GFP). Retrovirus-mediated labeling of dividing cells with GFP or other modified fluorescent proteins enables the direct visualization of whole cell morphology in live brain slices, which has led to significant advancements in our understanding of the unique electrophysiological properties of newborn granule cells. Furthermore, because the recombinant virus genome is integrated into the host chromosome, labeled cells can be traced at any stage after infection (reviewed in Zhao, 2008). In addition to BrdU and retrovirus vectors, transgenic mouse models have been generated to identify newborn neurons at different stages. For example, proopiomelanocortin (POMC) and DCX-dsRed2 mouse lines enable the visualization of immature neurons (CouillardDespres et al., 2006; Overstreet-Wadiche et al., 2005). The Nestin-creER mouse line permits the permanent

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labeling of newborn neurons in reporter mouse lines, such as Rosa26-YFP (Lagace et al., 2007). Thus, each of these techniques has pros and cons that complement each other: the BrdU approach is minimally invasive and labels most dividing cells but is restricted to the nucleus and requires tissue fixation before visualization; the transgenic approach is also noninvasive and does not require tissue fixation, but it allows the visualization of newborn neurons at only an immature stage; the viral approach is more invasive, labels a subset of dividing cells but labels the whole cell body, and enables the analyses of synapse formation on adult-born neurons and those requiring live tissue, such as electrophysiology.

38.2 SYNAPTOGENESIS IN THE ADULT HIPPOCAMPUS – NEWBORN GRANULE CELLS 38.2.1 An Overview of the Maturation Process of Newborn Granule Cells Mature granule cells extend dendrites in the molecular layer of the DG, where they receive axo-dendritic GABAergic input from local interneurons, mainly in the inner third of the molecular layer, and axo-spinous glutamatergic input from the entorhinal cortex, mainly in the outer two-thirds of the molecular layer. Other inputs include axo-somatic GABAergic input from hilar

interneurons, glutamatergic input from hilar mossy cells, cholinergic and GABAergic input from the medial septal nucleus and from the diagonal band of Broca, input from the supramammilary area, noradrenergic input from the locus coeruleus, serotoninergic input from the raphe nucleus, and dopaminergic input from the ventral tegmental area (reviewed in Amaral et al., 2007). As for their output, granule cells project to the hilus and the CA3 area, where they synapse with interneurons or pyramidal neurons, respectively (Claiborne et al., 1986, reviewed in Henze et al., 2000). New cells in the adult DG form synapses with the same input and output cells as existing mature granule cells (Laplagne et al., 2006, 2007). In fact, mature adultborn granule cells do not differ from existing cells in their electrophysiological properties or their connectivity. However, before they reach maturity, adult-born cells go through distinct stages of morphological and electrophysiological development (Figure 38.2). New neurons in the DG derive from hippocampal progenitors. Two types of progenitors exist in the DG: type 1 and type 2 cells. They differ in morphology and in the expression of certain molecular markers (reviewed in Kempermann et al., 2004; Zhao et al., 2008). Briefly, type 1 cells are relatively quiescent. They have radial processes that project through the GCL and ramify in the inner molecular layer. Type 2 cells are more proliferative and have short processes. Both type 1 and type 2 cells express stem cell markers, such as Nestin and

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GABA input tonic GABA input dendritic Glutamate input spine Glutamate output hilus Glutamate output CA3 GABA input soma

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FIGURE 38.2 Timeline of adult-born neuron development showing typical morphology, receptors expression, and synapse formation at different ages.

38.2 SYNAPTOGENESIS IN THE ADULT HIPPOCAMPUS – NEWBORN GRANULE CELLS

Sox2. Type 2 cells can be further categorized into 2a and 2b cells, with type 2b cells starting to express immature neuronal markers such as doublecortin and PSA-NCAM (Kronenberg et al., 2003). While it is debatable whether type 1 cells express receptors for major neurotransmitters; most of the PSA-NCAM þ type 2b cells identified in the NestinGFP transgenic mice express gamma-aminobutyric acid (GABA) receptors, and some appear to receive GABAergic synaptic inputs (Tozuka et al., 2005; Wang et al., 2005). However, when the birth date is identified by retrovirus labeling, newborn neurons of 1 week of age or younger express receptors for the major neurotransmitters GABA and glutamate, but they do not display afferent synaptic connections (Esposito et al., 2005; Ge et al., 2006). Given that these studies all used the adult mouse as a model system, one possible explanation of this discrepancy is that some of the Nestin-GFP þ PSA-NCAM þ type 2b cells may be older and thus more mature than the 1-week-old cells identified by retrovirus labeling (Fukuda et al., 2003). During the second week, new neurons extend axonal and dendritic processes concomitantly and receive mainly depolarizing synaptic GABAergic input. During the third week after cell birth, new neurons continue to expand their dendritic and axonal processes. Efferent synapses formed by the mossy fiber axons of newborn cells can be detected as early as 2 weeks after cell birth (Faulkner et al., 2008). Glutamatergic synaptic inputs are first detected at 18 days, just 2 days after the first dendritic spines are formed (Esposito et al., 2005; Zhao et al., 2006). Concomitantly, GABAergic input to newborn cells switches from depolarizing to hyperpolarizing (Ge et al., 2006). Between week 3 and week 6, newborn granule cells go through the peak of spine growth and maturation. Most importantly, cells at this stage have a low threshold for longterm potentiation (LTP) induction and higher amplitude of LTP. They may play a unique role that cannot be substituted by mature cells in the adult brain (Deng et al., 2009; Ge et al., 2007; Mongiat et al., 2009; Schmidt-Hieber et al., 2004). At that age, their connectivity is completed with the onset of perisomatic, fast GABAergic synapses (Esposito et al., 2005). New neurons 6–8 weeks of age do not display significant morphological differences from mature cells, but their dendritic spines and mossy fiber boutons are continuously modified (Faulkner et al., 2008; Toni et al., 2007, 2008; Zhao et al., 2006). After this period, these cells display electrophysiological properties similar to those of cells born during development (Laplagne et al., 2006). Thus, adult-born neurons integrate into the hippocampal circuitry, and all the studies so far indicate that their connectivity resembles that of prenatally born neurons. As detailed below, extended studies of GABAergic and glutamatergic connectivity have revealed traits that

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are unique to adult-born neurons and have relevance for their possible function.

38.2.2 Synaptogenesis on Dendritic Spines of Newborn Granule Cells As described above, the observation of dendrites from adult-born neurons was made possible by the viralmediated gene transfer of fluorescent proteins (van Praag et al., 2002; Zhao et al., 2006). With time after cell division in the mouse DG, dendrites of newborn granule cells grow into the molecular layer, reaching the inner molecular layer at 10 days and the middle molecular layer at 16 days. They reach their final size at around 4 weeks, although further dendritic growth still occurs between 4 and 8 weeks (Esposito et al., 2005; Ge et al., 2008; Overstreet-Wadiche et al., 2005; Zhao et al., 2006). Dendritic protrusions appear at around 16 days, and most spines are created during the third and fourth weeks, although spine density still increases until about 2–6 months after cell division (Toni et al., 2007; Zhao et al., 2006). In a manner similar to prenatal development, the early protrusions are thin, long, and very motile, probably reflecting immature filopodia and thin spines. The proportion of large, mature mushroom spines dramatically increases between 21 and 28 days and increases thereafter until at least 4 months, indicating long periods of remodeling and the stabilization of the circuitry. Using serial-section electron microscopy and three-dimensional reconstructions of dendritic protrusions, Toni et al. found that mature synapses impinge onto dendritic spines and include presynaptic vesicles, a synaptic cleft, and a long and clearly defined postsynaptic density. Using tracing experiments, dendritic spines from adult-born neurons are seen to contact perforant path axonal boutons (likely synapsing with them), suggesting that they receive glutamatergic input from appropriate anatomical afferences (Toni et al., 2007). However, critical questions are raised by the development of the connectivity of adult-born neurons. How does the incorporation of new neurons into the adult hippocampus modulate the existing network? Do dendritic spines contact preexisting or newly formed axonal boutons? Although the full resolution of these questions requires time-lapse imaging, the early indications from electron microscopy observations (Toni et al., 2007) suggest that filopodia are immature spines that actively search out presynaptic partners with which to stabilize and form a mature synapse (Knott et al., 2006; Marrs et al., 2001; Yuste and Bonhoeffer, 2004). When three-dimensionally reconstructed, the tip of filopodia is always very close (within 50 nm) to axon terminals, all of which are already synapsing with another granule cell; this distance is much smaller than the distance that would be

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expected if filopodia grew randomly in the neuropile. These observations suggest that filopodia are attracted by preexisting boutons from mature synapses. This possibility is supported by the observation that more mature protrusions, such as thin spines, synapse with axon terminals already synapsing with other neurons, thereby forming multiple-synapse boutons. In fact, about twothirds of the dendritic spines formed by 1-month-old adult-born neurons contact multiple-synapse boutons, suggesting a preference for preexisting synapses. Furthermore, along the course of their maturation, this proportion drops and stabilizes to about one-third, suggesting a transformation of connectivity during the development of adult-born neurons. These observations led the authors to hypothesize that a synaptic competition occurs between adult-born neurons and neurons that are more mature (Toni et al., 2007, Figure 38.3), supporting the view that glutamatergic activity regulates new neuron integration and survival (Tashiro et al., 2006). Thus, synaptogenesis on dendritic spines of adult-born neurons seems to modulate the connectivity of preexisting neurons.

38.2.3 Synaptogenesis of Axons from Newborn Granule Cells The first evidence that adult-born granule cells project to the hilus and the CA3 area came from tracing experiments (Hastings and Gould, 1999; Hastings et al., 2002; Markakis and Gage, 1999). By injecting the retrograde tracer fluorogold in the rat CA3 area 9 weeks after a BrdU injection, Markakis et al. found numerous double-labeled cells, suggesting that newly divided cells

sprout axons into the CA3 that pick up the tracer and transport it retrogradely into the GCL. These cells also stain for the mature granule neuronal marker Calbindin-D28k and are surrounded by synaptophysin immunoreactive puncta, revealing their neuronal identity and suggesting that they also receive a synaptic input. By analyzing rats at different time points after BrdU injection, Hastings et al. showed that the proportion of BrdU-immunoreactive cells containing the retrograde tracer increases from 0% at 4 days after BrdU injection to 9% at 10 days and 46% at 17 days. This indicates that the tracer is transported following the maturation of the axon and that the axon starts sprouting between 4 and 10 days. These results were later confirmed using the viral transduction approach in mice, which showed that axons start developing into the hilus at around 7 days and reach the CA3 at around 12 days (Zhao et al., 2006). The combination of viral-mediated gene transfer and serial-section electron microscopy made it possible to determine whether these axons develop mature synapses with their target neurons (Faulkner et al., 2008; Toni et al., 2008). This technique indeed demonstrated that, in mice, axonal boutons form synapses en passant with postsynaptic partners in the hilus and the CA3. In the CA3, the first presynaptic boutons are formed at around 10 days, but show no synaptic contact. The first synaptic contacts are formed at 2 weeks and involve small boutons containing few vesicles and dendritic shafts lacking spines, and are therefore immature. These boutons further develop into mature mossy terminals between 4 and 8 weeks, when they contact complex thorny excrescences and synapse with several spines.

FIGURE 38.3 Input

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Illustration of the hypothetical sequence of events involved in the synaptic integration of adult-born neurons in the glutamatergic network. Upper panel (input): a filopodia of an adult-born neuron (green) is attracted by a preexisting synapse between an axonal bouton (blue) and another neuron (red). When the filopodia stabilizes and matures into a dendritic spine, a multiple-synapse bouton is formed. The spine from the adult-born neuron progressively increases in size, and the spine from the other neuron decreases until it retracts, transforming the multiple-synapse bouton into a single-synapse bouton. Lower panel (output): the synaptic output follows rules similar to those observed by the input. Upon reaching the CA3, mossy terminals of new neurons (green) contact dendrites of pyramidal cells (red). Then a thorny excrescence either grows into the terminal or is shared between two terminals. After 2 months, single terminals contact single thorny excrescences. In blue: a presynaptic bouton from a preexisting neuron.

38.2 SYNAPTOGENESIS IN THE ADULT HIPPOCAMPUS – NEWBORN GRANULE CELLS

Interestingly, this time course is very similar to the development of the dendritic spines, where mature spine density and morphology is reached between 1 and 2 months. Another similarity with the synaptic input is shown by the connectivity of the contacted targets. Indeed, when they start contacting complex thorny excrescences, axon terminals from adult-born neurons contact thorny excrescences that are sometimes already involved in a synapse with another granule cell. These shared synapses, which are analogous to the multiple-synapse boutons, are not found when adult-born neurons are 75 days or older, which suggests an intermediate step in synaptogenesis, during which adult-born neurons share their synaptic partners with more mature cells. Finally, the functional maturation of these axon terminals was tested by electrophysiology. Due to their great length, mossy fibers are rarely maintained intact after the hippocampal slicing necessary for recordings. Therefore, these cells were transduced with the light-gated cation channel channelrhodopsin-2. In these conditions, a prolonged light stimulus induced glutamate-dependent postsynaptic responses in hilar interneurons and CA3 pyramidal cells, showing that adult-born neurons make functional synapses with appropriate postsynaptic partners (Toni et al., 2008). Thus, with the concomitant development of their synaptic input and output, adult-born neurons integrate into the hippocampal circuitry and develop a network similar to the network of neurons born prenatally. During the sprouting of their axons and dendrites, it is likely that the synaptic network of adult-born neurons changes. Indeed, during a short period of time, dendrites are restricted to the inner molecular layer and, almost at the same time, axons are restricted to the hilus. These areas are densely populated with GABAergic interneurons such as basket cells (Amaral and Lavenex, 2006). Thus, synapses formed during the initial phase of synaptogenesis are likely to involve mainly interneurons, while as they mature, adult-born neurons are more likely to synapse with more diverse partners, including glutamatergic input from the entorhinal cortex and output to pyramidal cells in the CA3. Such a shift in connectivity may induce physiological modifications in adult-born neurons, with implications for their function. The observation of both afferent and efferent synaptogenesis on adult-born neurons suggests that newborn neurons preferentially contact preexisting synapses and modulate the connectivity of preexisting neurons. This raises two important and unanswered questions with regard to synaptogenesis: 1. Are multiple-synapse boutons a morphological correlate of synaptic competition occurring between adult-born neurons and neurons that are more mature?

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2. Is the formation of multiple-synapse boutons and their conversion into single-synapse boutons a general mechanism for synaptogenesis in the adult brain?

38.2.4 Regulation of Synaptogenesis by Experience and Disease Hippocampal neurogenesis can be regulated at various levels, from the proliferation of progenitors, to fate determination, to the survival of newly generated cells, which will lead to an increase or decrease in the total number of new neurons generated by adult neurogenesis (reviewed in Abrous et al., 2005; Ming and Song, 2005; Zhao et al., 2008). This inevitably influences how local circuitry is modified by adult neurogenesis, as different numbers of new synapses will be formed by these cells. It is not yet clear how this modifies the function of the DG. Recent experimental studies suggest that new neurons may be recruited to encode certain experiences. For example, enriched environment (EE) is a paradigm frequently used to enhance adult hippocampal neurogenesis. Rodents exposed to EE display increased cell survival, and this effect is most prominent among cells of 1–2 weeks during the exposure (Kempermann et al., 1997; Tashiro et al., 2007). Moreover, these cells appear to respond more readily to the reexposure of the environment than to a different experience, such as the Morris water maze (MWM) task (Tashiro et al., 2007), suggesting that these newborn cells may contribute to the encoding of the EE. This is consistent to the observations that immature neurons contribute to memory retention in the MWM task (Deng et al., 2009). It is not clear how environmental enrichment affects synapse formation onto newborn granule cells. We will focus here on how other experiences shape the integration of adult-born neurons. 38.2.4.1 Exercise Voluntary exercise is by far the most efficient and natural way to enhance hippocampal neurogenesis. This is correlated with increased LTP in DG in mouse brain slices and better performance of running mice in MWM task and a newly designed touch screen task for pattern separation (Creer et al., 2010; van Praag et al., 1999). Exercise does not appear to alter dendritic growth or branching, as analyzed by the morphology of DCX þ cells (Couillard-Despres et al., 2005; Plumpe et al., 2006). Nor does it have a significant effect on the density of dendritic spines in retrovirus-labeled adult-born neurons (Zhao et al., 2006). However, exercise appears to promote the maturation of spines, as the density of mushroom spines reaches its plateau faster in mice housed with running wheels than those housed in standard mouse cages. The functional role of these new

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synapses is unclear, but the enhanced maturation of dendritic spines would suggest that the synapses formed by newborn cells are strengthened. This is consistent with the notion that newborn granule cells may encode experiences that induce their maturation (Aimone et al., 2009; Tashiro et al., 2007). 38.2.4.2 Learning Both behavioral studies and computational modeling suggest that adult-born granule cells contribute to certain forms of learning and memory. Learning also directly affects the integration of newborn cells, again supporting the hypothesis that new neurons may encode certain experiences. Learning influences the survival of newborn cells in an age-dependent manner (Ambrogini et al., 2000, 2004; Dupret et al., 2007; Epp et al., 2007; Gould et al., 1999; Kempermann et al., 1997; Mohapel et al., 2006). In adult rats, learning promotes the survival of new cells that are born 1 week before the MWM training, but induces the death of new cells that are slightly younger (born 3 days before MWM training). Most interestingly, the death of the latter population may be critical for the learning-induced survival of the relatively older cells and necessary in order for the mice to perform well in the task (Dupret et al., 2007). It is not yet clear whether and how the selective survival and death of distinct populations of newborn granule cells contribute to hippocampaldependent learning and memory. Morphological analyses of DCX þ newborn cells showed that learning increased the dendritic complexity of the newborn cells born 1 week before training. The remaining cells that were born shortly before training also showed increased dendritic complexity (Tronel et al., 2010). The increased complexity of newborn cells appears to persist for months (Tronel et al., 2010). Although the survival effect of the MWM task is dependent on learning, the morphological effect of the task does not appear to be. Using retrovirus labeling to birthdate newborn cells, Ambrogini et al. showed that cells born in learning rats were also more complex than those born in home-cage control rats, but they did not differ morphologically from cells born in swimming rats or cued-test rats (Ambrogini et al., 2010). Therefore, it is not clear how much of the morphological changes observed can be attributed to the active learning of the MWM task. It is possible that the spatial exploration may play a more significant role in shaping the synaptic connections of newborn granule cells, as the swimming and cued-test groups go through similar spatial experiences as learning groups, and these synaptic modifications can be recruited to learn the location of the hidden platform when necessary. For example, such representation can be reinforced

through the regulation of cell survival by learning, but not by mere spatial exploration (as in the swimming or cued-test groups) (Dupret et al., 2007). 38.2.4.3 Antidepression Treatments Adult hippocampal neurogenesis may participate in mood regulations. This hypothesis originated from observations that hippocampal neurogenesis can be effectively promoted by antidepression treatments, including electroconvulsive treatments and chronic administration of antidepressants (Malberg et al., 2000, reviewed in Warner-Schmidt and Duman, 2006). Although the level of hippocampal neurogenesis is not significantly affected by depression itself, antidepression treatments in rodents consistently promote the proliferation in the DG and the generation of newborn granule cells. This is correlated in mice with an increase in DG plasticity as well as with improved behavior in tasks designed to assess mood and depression (Santarelli et al., 2003; Saxe et al., 2006; Wang et al., 2008). Moreover, neurogenesis in the DG may be required for the beneficial effect of antidepressants, as suggested by the observation that antidepressant treatments were no longer effective when adult neurogenesis is blocked by X-ray irradiation (Santarelli et al., 2003). Antidepression treatments may also alter the property of granule cells in the adult hippocampus, including both newborn and existing cells (Kobayashi et al., 2010; Wang et al., 2008). Chronic fluoxetin treatment stimulates the dendritic complexity of DCXþ cells, suggesting that it may promote the maturation of these cells. Interestingly, chronic fluoxetin administration appears to have the opposite effect on mature granule cells, which lose expression of certain mature granule-cell markers in response to the treatment. It is yet to be confirmed that mature cells revert to an immature stage. Nevertheless, neuronal properties of granule cells (young or old) change as a result of the antidepressant treatment, which will affect their connectivity in the local circuitry. 38.2.4.4 Epilepsy Most of the pathological conditions affect cell proliferation in the DG negatively, but seizures promote cell proliferation very effectively. This increase in cell proliferation does not appear to correlate with animals’ performances in learning tasks, probably due to other changes in the hippocampus such as the loss of interneurons (Buckmaster and Dudek, 1997). In addition to promoting cell proliferation, seizure also leads to drastic changes in the connectivity of newborn granule cells, as well as the connectivity of mature cells. The most striking abnormalities of newborn cells born after seizure include basal dendrite formation and ectopic migration, and these aberrant cells persist in the epileptic brain (Jessberger et al., 2007; Kron et al., 2010; Walter et al., 2007, reviewed in Jessberger and Parent,

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38.2 SYNAPTOGENESIS IN THE ADULT HIPPOCAMPUS – NEWBORN GRANULE CELLS

2008). Under normal conditions, granule cells project 1 or 2 primary dendrites through the GCL that ramify in the molecular layer. Short basal dendrites form on these cells, but only transiently, when these cells are immature. Also, newborn granule cells normally locate in the GCL bordering the hilus. In rodent models of epilepsy, granule cells that bear long spiny basal dendrites are frequently observed. In addition to migrating into the GCL, a small population of new cells also migrates into the hilus and remains there even after they mature. EM studies clearly show that these abnormal cells form synapses at these ectopic locations (Jessberger et al., 2007). Under normal conditions, the axons of granule cells project into the hilus and the CA3 area. In seizure animals, mossy fibers were frequently found in the inner molecular layer (a phenomenon known as mossy fiber sprouting). Mossy fiber sprouting occurs in neurons that are relatively mature (4 weeks or older). Interestingly, 4–6-week-old granule cells born in the epileptic brain display decreased excitability when compared to cells born in the hippocampus of running rats (Jakubs et al., 2006). Seizure can also promote functional integration of newborn granule cells, which may be partly responsible for the decreased excitability in 4–6-week-old cells, as new cells lose their high excitability after 6–7 weeks (Ge et al., 2007; Jakubs et al., 2006; Mongiat et al., 2009; Overstreet-Wadiche et al., 2006). Therefore, it is still an open question whether aberrant neurogenesis contributes to or mitigates the epileptogenic network dysfunction in seizure patients. 38.2.4.5 Aging and Neurodegenerative Diseases The decline in hippocampal neurogenesis with aging is correlated with deterioration in learning and memory in both humans and rodents. Since exercise and an EE are among the most efficient and natural ways to enhance hippocampal neurogenesis, they have been used to partially restore hippocampal neurogenesis in aging animals or in mutant mice of neurodegenerative models (Kempermann et al., 1998; Lazic et al., 2006; van Praag et al., 2005). In addition to decreased neurogenesis, aging also produced a delay in the development of new neurons (Lazic et al., 2006; Rao et al., 2006), which is partially rescued by exercise and enriched environment (EE) (Lazic et al., 2006; van Praag et al., 2005). Hippocampal neurogenesis is also affected in mouse models of neurodegenerative diseases. Both cell proliferation and neuronal integration can be affected in these disease models. For example, neurite outgrowth of newborn cells is significantly delayed in a mouse model of Huntington’s disease, and enriched-environment housing can partially compensate for this (Lazic et al., 2006). In mouse models of Alzheimer’s disease, newborn granule cells display decreased dendritic complexity

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when they mature, which may be caused by an irregular GABAergic network in the DG of the mutant mice (Li et al., 2009; Sun et al., 2009). In the next section, we will discuss the latter in more detail.

38.2.5 Signaling Molecules that Regulate Synaptogenesis of Newborn Granule Cells Neurotransmitters play a fundamental role in regulating adult neurogenesis in the DG, indicating the importance of neuronal activity to the modification of the local circuitry. Indeed, neurotransmitters such as GABA, glutamate, dopamine, serotonin, acetylcholine, norepinephrine, neuropeptide Y (NPY), pituitary adenylate cyclase-activating peptide (PACAP), or nitric oxide have an effect on the proliferation and/or the survival of adult-born neurons that ultimately influences the total number of cells added to the existing circuitry (reviewed in Ming and Song, 2005). We discuss here how the integration of newborn cells is regulated by neurotransmitters and by other molecules. 38.2.5.1 GABA GABA plays a pleiotropic role during the early stages of neuronal development. As discussed earlier, type 2 hippocampal progenitors may be innervated by GABAergic interneurons in the hilus. GABAergic stimulation promotes the expression of NeuroD in type 2 cells and their differentiation (Tozuka et al., 2005; Wang et al., 2005). Newborn granule cells in adult mice express receptors for GABA and are tonically activated by GABA as early as 3 days after cell birth (Esposito et al., 2005; Ge et al., 2006). As these cells grow, they start to form GABAergic synaptic connections. Because the [Cl] is high in immature granule cells, GABAergic input depolarizes newborn granule cells and is important for the dendritic development of newborn cells. When the Cl importer NKCC1 is decreased by shRNA, GABA stimulation leads to hyperpolarization in newborn cells, and this causes a severe delay in neuronal development (Ge et al., 2006). Consistently, NKCC1 knockdown cells display defects in the formation of both GABAergic and glutamatergic synapses (Ge et al., 2006). The trophic effects of GABA during early development may be mediated by cAMP response element-binding (CREB) protein (Jagasia et al., 2009). As discussed earlier, the connectivity of newborn granule cells is affected in two mouse models of Alzheimer’s disease, one with a transgenic line that expresses human amyloid precursor protein (hAPP-J20) and the other with a knock-in mouse line that expresses human ApoE proteins (Li et al., 2009; Schinder and Morgenstern, 2009; Sun et al., 2009). In the hAPP-J20 line, GABAergic fibers in the DG are dramatically

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increased in comparison to NPY, as determined by immunostaining (Sun et al., 2009), whereas the ApoE4KI mice display decreased GABAergic interneurons and GABA release in the DG (Li et al., 2009). Interestingly, newborn granule cells display similar morphological defects in these two mouse lines. Dendritic growth and complexity were reduced and spine density was lower. Moreover, newborn cells in hAPP-J20 mice appeared to develop faster initially. These data are consistent with the role of GABAergic inputs to promote dendritic development during early stages. The role of GABAergic inputs at later stages of granule cell development has not been determined yet. 38.2.5.2 Glutamate Newborn granule cells start to receive glutamatergic synaptic inputs during the third week after their birth, which is within the same time window when they start to form dendritic spines (Esposito et al., 2005; Zhao et al., 2006). They express glutamate receptors when they are much younger (Esposito et al., 2005), and glutamate indeed regulates cell proliferation: the activation of NMDA receptors reduces cell proliferation and NMDA antagonists induce cell proliferation (Cameron et al., 1995). In mice, the survival of newborn granule cells is highly dependent on the NMDA receptors during the period when they start to receive glutamatergic excitatory synaptic inputs, but not at earlier stages (Tashiro et al., 2006). NMDA receptor-dependent activity is also required for the increased dendritic complexity of newborn cells in response to learning. Interestingly, in rats this mainly affects cells that are 1–2 weeks old during the learning sessions (Tronel et al., 2010). At this time window, newborn cells express glutamate receptors, but are yet to form dendritic spines or glutamatergic synapses. Therefore, it is possible that the NMDA receptor play an extrasynaptic role at this early stage before excitatory synapses are formed on newborn neurons. 38.2.5.3 Other Neurotransmitters As mentioned above, adult-born granule cells are modulated by other neurotransmitters as well, but their connectivity has been not been as thoroughly investigated. Acetylcholine promotes neuronal stem or progenitor cell proliferation in vivo (Ide et al., 2008; Itou et al., 2011), possibly through direct synaptic contact from the medial septum cholinergic axons (Ide et al., 2008). The effect of dopamine on adult hippocampal neurogenesis has been indirectly studied with the use of cocaine, a serotonin–norepinephrine–dopamine reuptake inhibitor, which shows an inhibitory effect on cell proliferation (Dominguez-Escriba et al., 2006). Norepinephrine also promotes neurogenesis by stimulating stem/progenitor cells (Jhaveri et al., 2010). Finally, serotonin depletion in

the hippocampus decreases adult neurogenesis (Brezun and Daszuta, 1999), whereas the serotonin reuptake inhibitor fluoxetin increases the proliferation and maturation of adult-born neurons (Malberg et al., 2000; Wang et al., 2008). It is unclear, however, whether the effect of these neurotransmitters is produced by direct or indirect synapses. 38.2.5.4 Other Molecules that Regulate Synapse Formation of Newborn Granule Cells Adult-born granule cells are influenced by some common pathways that regulate the integration of neurons born during early development. For example, the reeler mice have severe defects in the hippocampus, especially in the DG, which is completely disorganized. The deficiency of Reelin may also be responsible for the ectopic migration of adult-born granule cells in the epileptic brain (Gong et al., 2007). Overexpression of Reelin induces an increase in hippocampal neurogenesis and the mispositioning of newborn granule cells (Pujadas et al., 2010). In some cases, certain molecules are so critical for neuronal development that it is difficult to study their precise function during early development. Adult neurogenesis provides a unique platform by which to bypass this problem, as one can specifically alter the expression of such genes in adult-born neurons without affecting other cells in the brain. For example, the deletion of the gene for cyclin-dependent kinase 5 (Cdk5) in mice leads to perinatal lethality, which prevents the assessment of its role in neuronal development and integration. Using retrovirus-mediated expression of a dominant negative form of Cdk5 or genetic mouse models to specifically delete the gene in progenitor cells in the adult brain, Jessberger et al. (2008) and Lagace et al. (2008) found that Cdk5 is required for the dendritic development and synapse formation of newborn granule cells. In addition, in a manner consistent with the role played by Cdk5 in neuronal migration during early development, Cdk5 is also critical for the correct positioning of adult-born neurons (Jessberger et al., 2008). The TrkB gene, which encodes the receptor for the neurotrophic factor BDNF, is important for the dendritic arborization and spine growth of newborn granule cells, and its presence in newborn granule cells may be involved in mood regulation (Bergami et al., 2008). In contrast to Reelin, Cdk5, and TrkB, DisruptedIn-Schizophrenia 1 (DISC1) appears to play a role in the adult-born neurons that is different from the role it plays in developmentally born neurons. Disruption of the gene encoding DISC1 has been suggested to contribute to the pathophysiology of schizophrenia, hence the nomenclature. During development, DISC1 promotes neuronal migration and neurite growth. In adult-born neurons, DISC1 plays an opposite role. By knocking down DISC1 expression using retrovirus-mediated

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38.3 SYNAPTOGENESIS IN THE ADULT HIPPOCAMPUS – CA1 AND CA3 PYRAMIDAL CELLS AND OTHER NEURONS OF THE HIPPOCAMPUS

expression of small hairpin RNA, Duan et al. showed that in the absence of DISC1 new neurons migrate further into the GCL and even into the molecular layer (Duan et al., 2007). Similarly, the mossy fiber axons of DISC1 negative cells migrate beyond the CA3/CA1 border, unlike those of wild-type cells, which always terminate at the CA3 area (Faulkner et al., 2008). In addition, the dendrites and axons of adult-born cells develop much faster in the absence of DISC1 (Duan et al., 2007; Faulkner et al., 2008). In adult-born granule cells, DISC1 may exert its influence through the AKT–mTOR signaling pathway, by binding to Gridin (Enomoto et al., 2009; Kim et al., 2009). Since decreased neurogenesis was observed in schizophrenic patients (Kempermann et al., 2008), it will be important to determine whether aberrant hippocampal neurogenesis contributes to the etiology of schizophrenia.

38.2.6 Theoretical and Computational Models of Synaptogenesis of Newborn Cells and Their Function in the Adult Hippocampal Neurogenesis As mentioned briefly earlier in this chapter, adult neurogenesis in the DG may contribute to certain forms of learning and memory. However, discrepancies exist among different animal models and different behavioral tasks. This is partially due to some methodological limitations. So far, most of the behavioral tasks employed examine the function of the hippocampus, of which the DG is only one part. Moreover, the precise role of the DG has not been investigated extensively. To overcome these limitations, several computational models have been established in order to examine the functional role of the DG as well as to determine how adult neurogenesis may contribute to its function. In general, the DG is thought to play a role of sparsification and pattern separation (reviewed in Aimone and Wiskott, 2008). This is based on two prominent features of the DG: (1) the number of granule cells greatly exceeds the number of its main input cells in the entorhinal cortex and (2) granule cells are highly innervated by local interneurons. There are two major types of computational modeling used to examine the potential function of adult neurogenesis in the DG: replacement models or additive models (reviewed in Aimone et al., 2010). Both types of modeling predict that neurogenesis is beneficial to the formation and/or retrieval of new memories. This is consistent with observations made in animal models, in which increased neurogenesis correlates with better performance in learning tasks. These models differ in their predictions regarding the issue of how old memories are influenced by neurogenesis. In general, replacement models tend to suggest that neurogenesis may promote the clearance of old memories, whereas

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additive models suggest that neurogenesis helps to preserve old representations. These different models have focused on different aspects of DG function, but they are all based on the pattern separation function proposed for the DG (McNaughton and Morris, 1987; Treves and Rolls, 1992). For example, Becker (2005) proposed that neurogenesis contributes to the formation of distinct memory traces for highly similar inputs. The modeling work by Weisz and Argibay (2009) suggested that neurogenesis contributes to the encoding/retrieval of recent memories, but does not contribute significantly to the encoding/retrieval of more remote memories. This differs slightly from the prediction of Appleby and Wiskott (2009) that neurogenesis helps to preserve representations of earlier environments. Taking into consideration the dynamic changes of neuronal properties during their maturation, Aimone et al. (2009) proposed different functions for newborn cells based on their developmental stage. Immature, highly excitable newborn granule cells contribute to the pattern integration function of the DG, which facilitates the association of dissimilar inputs within a short time frame. Over time, continuous neurogenesis throughout life creates a turnover of this immature population, which contributes to the temporal separation of distinct events. Moreover, newborn cells appear to respond specifically to the environment in which they matured, suggesting that they may contribute to the encoding of familiar events. These computational studies will no doubt be instrumental in guiding designs of new behavior paradigms to pinpoint the precise function of adult hippocampal neurogenesis.

38.3 SYNAPTOGENESIS IN THE ADULT HIPPOCAMPUS – CA1 AND CA3 PYRAMIDAL CELLS AND OTHER NEURONS OF THE HIPPOCAMPUS In the hippocampus, adult neurogenesis is restricted to granule cells, and the synaptic network is thus expected to be more stable in the Ammon’s horn than in the DG. However, while adult-born neurons extend their dendrites in the molecular layer and their axons in the CA3 area, the cells to which they connect undergo dramatic connectivity rearrangement that involves synaptogenesis (see Section 38.2.3). The study of adult-born neurons is greatly facilitated by the design of viral transduction approaches, which enable both the identification of transduced cells in living tissue and a relatively accurate birth dating. Such viral approaches do not, however, apply to neurons born prenatally in other parts of the hippocampus. Furthermore, the observation of synaptogenesis in the adult hippocampus is technically challenging. Indeed, the anatomical

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localization of the hippocampus in subcortical areas and its lamellar organization cause in vivo morphological observations to be very invasive and incomplete. In addition, the survival of organotypic slice cultures prepared from adult animals is limited and hinders observations over long periods of time, which may be required for studying relatively discrete events. Therefore, many studies of synaptogenesis have been performed on organotypic slices prepared from early postnatal ages, and it is assumed that the consequent observations can be extrapolated on adult hippocampus. Using the postnatal organotypic slice culture system, it was observed that the induction of LTP (a physiological mechanism for learning and memory) in the Schaffer collateral/CA1 synapses stimulates morphological modifications of the activated synapses, leading to dendritic spine growth on CA1 pyramidal neurons, postsynaptic density perforation, and new spine formation (Engert and Bonhoeffer, 1999; Toni et al., 2001). When reconstructed three-dimensionally, these dendritic spines were observed to grow very close to the activated synapse and to contact the same presynaptic terminal, thereby forming multiple-synapse boutons (Toni et al., 1999). Thus, multiple-synapse boutons may indeed be a morphological correlate for synaptogenesis in the mature hippocampus. As opposed to the multiple-synapse boutons observed during neurogenesis, which were formed by one presynaptic and two different postsynaptic cells and therefore lead to a divergent signal, multiplesynapse boutons formed by LTP induction are formed by dendritic spines stemming from the same postsynaptic neurons. As such, they lead to a convergent signal, thereby strengthening the connectivity between two activated neurons. The possibility that multiple-synapse boutons formation is a morphological correlate of synaptogenesis in the adult brain is supported by observations that estrogen increases spine density and synaptogenesis on CA1 neurons in female rats and results in the formation of multiple-synapse boutons with multiple postsynaptic neurons (Yankova et al., 2001). The mechanisms of multiple-synapse bouton formation are currently unknown, but one possibility is that glutamate spillover from preexisting synapses attracts nascent dendritic spines. In support of this possibility stands the observation of dendritic spine growth. Dendritic spine formation is regulated by several factors, among which glutamate plays a major role. Indeed, dendritic spine formation is triggered by exogenous glutamate application, and the spine heads show a clear directionality toward the glutamate electrode (Richards et al., 2005). This suggests that spontaneously released glutamate may be sufficient to activate nearby spines and that this tropism may induce the abovementioned preference of newborn neurons for preexisting synapses and the subsequent formation of multiple-synapse boutons.

The remarkable plasticity of dendritic spines is a prerequisite for synaptogenesis, but presynaptic terminals undergo major structural changes, as well. Although presynaptic plasticity has been less studied, the formation of multiple-synapse boutons induces the rearrangement of docked presynaptic vesicles (Toni et al., 1999, 2001, 2007), and axonal branches and presynaptic terminals also show structural plasticity involving synaptogenesis (Gogolla et al., 2007). In the mossy fiber–CA3 synapses, presynaptic plasticity and synaptogenesis are regulated by neuronal activity and neurotransmitter release (Galimberti et al., 2006), suggesting the existence of a transsynaptic control of events during synapse formation.

38.4 CONCLUSIONS Adult neurogenesis represents a unique form of plasticity in the hippocampus. In other hippocampal areas, the mechanisms of learning and memory involve synapse remodeling, formation, and elimination, but not the addition of new neurons, as this dramatic form of plasticity is unique to the DG. The total amount of synapses formed by this process is much greater than that resulting from individual synaptic plasticity, and it is likely that the timing of the insertion of these neurons determines their encoding. But what is the function of adult hippocampal neurogenesis? Is the network remodeling induced by the addition of neurons functionally different from the addition of discrete synapses? If so, how is it different? The answers to these questions rely on a better understanding on the mechanisms of synaptic plasticity presented by adult neurogenesis. Both the EM studies discussed above and electrophysiological recordings of newborn granule cells clearly demonstrate that adult-born granule neurons integrate into the DG circuitry, forming synaptic contacts as early as 1–2 weeks. Their dynamic properties change during maturation, suggesting that these cells play distinct roles at different stages. This idea has been supported by the observation that mice display defects in trace fear conditioning tasks or long-term memory in the traditional MWM task only when the immature population is ablated or reduced (Deng et al., 2009; Shors et al., 2001). It is also consistent with computational predictions that immature neurons help to associate events that occur within a short time window (Aimone et al., 2009). However, it is not clear how, mechanistically speaking, these young, immature neurons affect the circuitry. Given that the synapses formed by these cells are very immature at this early stage, it is critical to determine how these immature synapses affect network activity.

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38.4 CONCLUSIONS

A second important question regarding synaptogenesis on newborn granule cells is this: what is the function of newborn cells after they pass the early immature stage? New neurons are still quite distinct from mature cells when they are 4–6 weeks old. They display significantly enhanced LTP and remain highly excitable. Moreover, new synapses are still formed on these neurons, and existing synapses are continuously modified. It has been suggested that new neurons may encode information about the experience in which they matured, as they can be preferentially activated by the same experience over a different experience. However, only a fraction of cells are activated in such cases, and mature cells can be activated at a similar rate, suggesting that while a small ensemble of newborn cells are recruited to encode past representation, the rest of them may still be recruited by new experiences. The EM observations about multiple-synapse boutons raise a third question: does synapse competition result in the turnover of synapses or in the splitting of synapses? This is analogous to the unresolved question as to whether adult neurogenesis in the DG leads to neuronal turnover or replacement. Clearly, these two different possibilities will determine whether synapse formation by newborn neurons interferes with old network representations. We are hopeful that, with the advancement of in vivo imaging techniques, this question can be answered in the not-too-distant future.

SEE ALSO Synaptogenesis: Molecular Composition of Developing Glutamatergic Synapses; In Vivo Analysis of Synaptogenesis; Activity-regulated Genes and Synaptic Plasticity; New Imaging Tools to Study Synaptogenesis; Synaptogenesis in the Adult CNS – Cortical Plasticity.

Acknowledgments We thank Drs. James B. Aimone and Alejandro Schinder for helpful discussions, Jamie Simon for illustration, and Mary Lynn Gage for editorial comments. We also acknowledge the support of the Lookout Fund, the Defense Advanced Research Projects Agency (DARPA), the U.S. National Institutes of Health (NS-05050217, NS-05052842 and MH090258), and the National Institutes of Aging (AG-020938), the James S McDonnell Foundation and the Mathers Foundation to F.H.G., and the Swiss National Science Foundation to N.T.

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