University of Groningen. New neurons in the adult brain van der Borght, Karin

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1 University of Groningen New neurons in the adult brain van der Borght, Karin IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2006 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): van der Borght, K. (2006). New neurons in the adult brain: A study on the regulation and function of neurogenesis in the adult rodent hippocampus. s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 Chapter 1 General Introduction 9

3 Chapter 1 CONTENTS 1 History of neurogenesis research 2 The dentate gyrus 2.1 Anatomy 2.2 Input pathways to the DG 2.3 The septohippocampal pathway 3 Hippocampal neurogenesis: from stem cell to functional granule neuron 3.1 The putative hippocampal stem cell 3.2 Different subsets of proliferating precursor cells 3.3 Maturation of the newly formed cells 3.4 Fluctuations in newly formed cell number 4 Regulation of hippocampal neurogenesis 4.1 The neurogenic microenvironment 4.2 Internal and external factors that regulate hippocampal neurogenesis 4.3 Effects of physical exercise on hippocampal neurogenesis 5 Neurogenesis and learning 5.1 Environmental impact on hippocampal neurogenesis 5.2 Effects of learning on neurogenesis 5.3 Effects of reduced neurogenesis on learning 5.4 Computational models of neurogenesis: neurogenesis and memory clearance 6 Outline of this thesis 10

4 General introduction 1. History of neurogenesis research For over 100 years, the generally accepted view has been that the adult brain only contains postmitotic neurons and is incapable of generating new ones (Gross, 2000; Ramon y Cajal, 1928). The introduction of [3H]-thymidine, which is incorporated in the DNA of mitotic cells during the S-phase of the cell cycle, resulted in the discovery of newly formed cells in the adult rat brain (Altman and Das, 1965). These fi ndings have been largely neglected for many years, mainly because the available techniques at that time could not indisputably demonstrate that the labeled cells were indeed new neurons. In the 1980s, electron microscopical studies demonstrated that newly formed cells in the dentate gyrus (DG) and the olfactory bulb had the ultrastructural characteristics of new neurons (Kaplan and Hinds, 1977). A few years later, the presence of neurogenesis in the vocal control nucleus of canaries was discovered (Goldman and Nottebohm, 1983). Interest in the neurogenesis field started to increase dramatically when studies in male canaries demonstrated that cell death and cell recruitment in the vocal nuclei are seasonally regulated and play a role in song modification (Kirn et al., 1994). The introduction of 5-bromo-2 -deoxyuridine (BrdU) as a tool to study the generation of new cells caused a rapid expansion of the fi eld. BrdU is a thymidine analogue, which is incorporated in replicating DNA, and in contrast to [3H]-thymidine, BrdU can be visualized with immunocytochemistry. In addition, the use of BrdU as a tool to label dividing cells makes it possible to investigate the phenotype of newly formed cells with double labeling techniques (Gross, 2000). With these new techniques, adult hippocampal neurogenesis could be demonstrated in many species, including primates (Gould et al., 1999b; Kornack and Rakic, 1999) and even humans (Eriksson et al., 1998). Adult neurogenesis has been identified in the granule cell layer (GCL) of the hippocampus and in the olfactory bulb. New olfactory bulb neurons originate from undifferentiated progenitors that are located along the wall of the lateral ventricles, the subventricular zone (SVZ). Upon migration towards the olfactory bulb, via the so-called rostral migratory stream (RMS), they differentiate into interneurons (Bedard and Parent, 2004; Winner et al., 2002). In the hippocampus, an undifferentiated, proliferating cell population is located in the small layer between the GCL and the hilus, the subgranular zone (SGZ). After a few cell divisions, the proliferating cells become postmitotic, migrate into the granule cell layer, differentiate into neurons and become integrated into the existing hippocampal circuitry. The focus of this thesis is on hippocampal neurogenesis. Therefore, whenever the term adult neurogenesis is mentioned, this refers to neurogenesis in the adult DG. 11

5 Chapter 1 2. The dentate gyrus 2.1. Anatomy The dentate gyrus (DG) consists of a granule cell layer, containing densely packed granule cells (circa 1,000,000 in rats (Boss et al., 1985) and 250, ,000 granule cells in mice (Kempermann et al., 1997; Sun et al., 2004)), a molecular layer, in which the granule cell dendrites are located and a polymorphic layer or hilus that contains the axons of the granule cells, various types of interneurons and astrocytes. The bundle of axons from the granule cells that projects to the CA3 region is called the mossy fi ber pathway. Mossy fiber axons innervate two different cell types in the CA3 region, the pyramidal neurons (around 300,000 in rats (Amaral et al., 1990)) and interneurons, via morphologically distinct presynaptic terminals. About 50 granule neurons project to one CA3 pyramidal cell and around 14 pyramidal cells are innervated by one granule neuron via a specialized giant mossy fi ber bouton (Amaral and Dent, 1981). Each granule cell also innervates 40 to 50 CA3 interneurons via fi lopodial extensions that extend from the giant bouton and en passant boutons along the main mossy fi ber axon (Acsady et al., 1998; Frotscher et al., 1994). Therefore, granule cells actually innervate more interneurons than principal CA3 cells, suggesting that GABAergic cells are the major postsynaptic target of granule cells. However, although not many pyramidal neurons are activated by a single granule neuron, the connection appears to be strong enough to function as a detonator or teacher synapse for the storage of information in the CA3 network (Henze et al., 2002; Jung and McNaughton, 1993; Lisman, 1999). Each mossy fi ber axon also makes approximately synapses with cells of the hilus. The hilar cells are primarily inhibitory interneurons, but the mossy fi ber collaterals of a single granule cell make approximately 10 synapses with excitatory hilar mossy cells that project back to the granule cells forming a dense plexus in the inner molecular layer of the dentate gyrus (Amaral, 1978; Buckmaster et al., 1996; Henze et al., 2000). The GCL can be subdivided into the suprapyramidal (inner) blade and the infrapyramidal (outer) blade. During development of the DG, which takes place postnatally in rodents, cells of the infrapyramidal blade are generated somewhat later than those of the suprapyramidal layer (Schlessinger et al., 1975). Morphology of granule cells differs between both blades (Claiborne et al., 1990), with cells in the suprapyramidal blade having more total dendritic length, more dendritic segments and a greater transverse spread. Spine density of dendritic spines is greater on suprapyramidal cells than on infrapyramidal cells (Desmond and Levy, 1985). Combined with their greater dendritic length, these numbers indicate that suprapyramidal cells receive a signifi cantly greater number of terminals. On the other hand, stimulation of the molecular layer causes a larger population spike in the infrapyramidal blade than in the suprapyramidal blade and the suprapyramidal blade requires higher stimulus intensities to evoke EPSPs that reach the threshold for action potential generation (Scharfman et al., 2002) 12

6 General introduction 2.2. Input pathways to the DG The DG receives input from a variety of brain regions, a few of which are shortly being discussed here. The major cortical innervation of the hippocampus comes from the entorhinal cortex (EC). The axons of the cells that are located in layer II of the EC project axons to the outer two thirds of the molecular layer. These projections form the so-called perforant path. The EC-DG projections are not homogenously distributed over the septo-temporal pole of the hippocampus. Cell from the lateral EC innervate the septal part, whereas cells in the medial EC connect to the temporal pole of the dentate gyrus (Dolorfo and Amaral, 1998; Hjorth-Simonsen and Jeune, 1972). The inner third of the molecular layer is occupied by associational and commissural fi bers, which are intrinsic fi ber systems of the hippocampal formation (Amaral and Witter, 1989; Deller, 1998). The DG also receives dense monoaminergic input. Serotonergic neurons in the medial raphe innervate the hippocampus via the supracallosal pathway through the fi m- bria-fornix and via the infracallosal pathway through the cingulate bundle. Axons from the dorsal raphe nucleus reach the hippocampus via the supracallosal pathway or through the amygdala and the EC (Azmitia and Segal, 1978; Gulyas et al., 1999; Kohler et al., 1982). In addition, the DG shows strong immunoreactivity for tyrosine hydroxylase (TH), the rate-limiting enzyme for noradrenaline and dopamine synthesis. However, the source of TH that is present in the DG predominantly appears to originate from noradrenergic neurons in the locus coeruleus (Ishida et al., 2000; Moore and Bloom, 1979), and not from dopaminergic neurons (Gasbarri et al., 1994; Torack and Miller, 1994) The septohippocampal pathway One of the major afferent connections to the hippocampus is formed by the medial septum (MS). The fi bers that originate in the MS enter the hippocampus via the fi mbria-fornix and form a diffuse network in all hippocampal layers. Whereas the perforant path is thought to relay specific sensory cortical information about the environment to the hippocampus, the input from the MS is considered to be involved in providing information about the motivational, emotional and autonomic state of the animal (Gulyas et al., 1999). The MS consists of three cell types: cholinergic neurons, GABAergic projection neurons and GABAergic interneurons. The cholinergic neurons in the MS innervate all three cell types in the hippocampus: the pyramidal cells, granule neurons and interneurons (Frotscher, 1989; Gaykema et al., 1990). The GABAergic projection neurons also send axons to the hippocampal formation, where they make contact with interneurons (Freund, 1992; Frotscher et al., 1992; Gulyas et al., 1999). Electrophysiological studies indicate that the cholinergic input to the dentate granule neurons causes a direct excitation of these cells (Hortnagl and Hellweg, 1997; Wheal and Miller, 1980). The cholinergic projection to hippocampal interneurons, leads to an increased interneuronal activity (Frazier et al., 2003; Pitler and Alger, 1992). Also within the MS, there is a complex communication between the different cell types. Not only do the interneurons inhibit the cholinergic neurons, the cholinergic 13

7 Chapter 1 neurons also exert an effect on the GABAergic neurons, which have been shown to express muscarinic receptors (Brauer et al., 1998; Van der Zee and Luiten, 1994). The cholinergic cells in the MS also express muscarinic receptors, but in a lower density than GABAergic neurons (Van der Zee and Luiten, 1994). A subpopulation of cholinergic neurons is inhibited by muscarinic stimulation, suggesting the presence of the inhibitory m2 receptor subtype (Wu et al., 2000). Finally, the hippocampus also has reciprocal connections with the MS. These projections come either from hippocampal pyramidal cells that innervate cholinergic neurons in the MS via the lateral septum (Leranth and Frotscher, 1989; Risold and Swanson, 1997), or from GABAergic, primarily somatostatin-positive neurons (Jinno and Kosaka, 2002) that directly project to GABAergic neurons in the medial septum and, to a smaller extent, also to cholinergic cells (Gaykema et al., 1991; Schwerdtfeger and Buhl, 1986; Toth et al., 1993) 3. Hippocampal neurogenesis: from stem cell to functional granule neuron 3.1. The putative hippocampal stem cell Neurogenesis is thought to depend on the presence of neural stem cells (NSC s). Stem cells can be defi ned as undifferentiated cells that show asymmetric cell division, that have the capacity to self-renew and that are multipotent. Since no specific marker for NSC s has been identified yet, an NSC is usually defi ned as a cell that has the capacity to form multipotent neurospheres in vitro. Based on this criterion, it has been questioned whether the adult hippocampal formation does in fact contain NSC s, since there are reports showing that clonogenic spheres are only formed in cultures of the SVZ and not of the hippocampus (Seaberg and van der Kooy, 2002). Others, however, were able to obtain multipotent neurospheres from the adult hippocampus (Gage et al., 1995; Palmer et al., 1999). Moreover, the fact that new neurons and glia cells continue to be produced throughout an animal s life suggests the presence of NSC s in the hippocampus (Alvarez-Buylla et al., 2002). Detailed analysis of the subventricular germinal zone demonstrated that the putative stem cell in this region is likely to be GFAP-positive (Alvarez-Buylla et al., 2002; Seri et al., 2001). There is also increasing evidence that the hippocampal stem cell is a GFAPexpressing cell (Garcia et al., 2004; Kempermann et al., 2004; Seri et al., 2001; Seri et al., 2004). In the SGZ two different types of GFAP-positive cells can be found, horizontal and radial astrocytes (Seri et al., 2001; Seri et al., 2004). The radial astrocytes or type-1 cells (Fukuda et al., 2003; Kempermann et al., 2004) are, in contrast to the multipolar appearance of horizontal astrocytes, predominantly uni-or bipolar and they have a radial process that goes through the granule cell layer and short tangential processes extending along the SGZ (Alvarez-Buylla et al., 2002; Fukuda et al., 2003; Garcia et al., 2004). Also at the electronmicroscopical level, differences can be observed between radial and 14

8 General introduction horizontal astrocytes. Radial astrocytes have lighter mitochondria, more organelles, more polyribosomes and more prominent intermediate fi lament bundles in their processes (Seri et al., 2001). Next to GFAP, the radial astrocytes express vimentin (Garcia et al., 2004) and nestin (Fukuda et al., 2003; Kempermann et al., 2004; Mignone et al., 2004; Zhuo et al., 2001), which are not expressed by differentiated multipolar astrocytes. Radial astrocytes actually make up about two-thirds of the nestin-expressing population of cells in the SGZ. However, of the total nestin-expressing population, radial glial like cells are thought to form only 5% of the dividing cells (Kempermann et al., 2004). Evidence that radial astrocytes in the SGZ are indeed the precursor cells for granule neurons comes from studies in which SGZ astrocytes were retrovirally labeled. A few weeks after labeling, positive granule neurons in the GCL were observed, indicating that these new neurons were derived from GFAP-positive cells (Seri et al., 2001). Some astrocytes remained labeled, suggesting symmetric cell division into two astrocytes or asymmetric cell division, a key feature of stem cells (Alvarez-Buylla et al., 2002; Steiner et al., 2004). Additional evidence supporting the theory that NSC s in the hippocampus express GFAP comes from studies with a transgenic mouse model in which the proliferating population of GFAP-expressing cells in the forebrain could specifically and conditionally be deleted. Elimination of this cell population entirely prevented the formation of new hippocampal neurons (Garcia et al., 2004). Using GFAP-Cre reporter mice, the fate of GFAP-progenitors was identifi ed. Two weeks after injection with BrdU, over 90% of NeuN-expressing BrdU-positive cells expressed the reporter protein, indicating that the majority of the newly formed neurons in the DG are derived from GFAP-expressing progenitors Different subsets of precursor cells Radial astrocytes do not directly differentiate into mature granule neurons, but fi rst give rise to different subtypes of intermediate precursors. Based on their morphology (Fukuda et al., 2003; Seri et al., 2001; Seri et al., 2004), their marker expression (Fukuda et al., 2003; Jessberger and Kempermann, 2003; Kempermann et al., 2004; Kronenberg et al., 2003; Steiner et al., 2004) and their electrophysiological properties (Ambrogini et al., 2004a; Fukuda et al., 2003) various subtypes of precursors have been described, thereby using different nomenclature. A summary of the different precursor subtypes that can be found in the adult hippocampus is presented in Figure 1 (page 138). Astrocytic stem cells give rise to rapidly proliferating cells, which are called D- cells by Seri and colleagues (Seri et al., 2001; Seri et al., 2004). These cells have a completely different morphology than radial astrocytes. They have smooth plasma membranes, dark cytoplasm, many polyribosomes (more than astrocytes, less than granule cells), lighter mitochondria than astrocytes and an endoplasmatic reticulum that is larger than in astrocytes, but smaller than in granule cells. Three subtypes of D cells have been defi ned: D1, D2 and D3. D1 cells are small, mitotic cells with little cytoplasm and no extensions (Seri et al., 2004) or only a very thin process (Fukuda et al., 2003). D2 cells have a short thick process and D3 cells have characteristics of immature neurons, which are frequently branched 15

9 Chapter 1 dendrites that reach through the GCL into the molecular layer and a thin process projecting into the hilus. D2 and D3 cells are postmitotic (Seri et al., 2004). D cells stain positive with PSA-NCAM, doublecortin (DCX) and TUC-4, which are all proteins involved in neurite outgrowth, and for NeuroD, a transcription factor involved in neuronal differentiation. biii-tubulin, a marker for mature neurons, was only found in some D cells. Kempermann et al. (2004) use a different nomenclature and, based on different criteria, further distribute the proliferating precursor population into various subtypes. First, as described earlier, there is the type 1 GFAP positive cell that resembles the radial glia cell that can be found during development. According to his model, the remaining GFAP-negative cells of the proliferating population of precursors can be subdivided into type 2A (nestin-positive, DCX-negative), type 2B (nestin-positive and DCX-positive) and type 3 (nestin-negative, DCX-positive) cells. Type 3 cells are also positive for PSA-NCAM, though NCAM polysialylation also occurs frequently in type 2B cells (Kronenberg et al., 2003). Type 2 and type 3 cells look largely similar, but during the type2/type3 stage, cells undergo great morphological changes. Finally, maturation state of the different precursors can be defi ned based on their electrophysiological properties. Fukuda and colleagues (2003) showed that cells that express both nestin and PSA-NCAM (named type 2B by Kempermann et al. and type II by Fukuda et al.), can be distinguished from other cell types in the GCL on the basis of their high input resistance. Compared to adult mice (388 MΩ) (Van Praag et al., 2002), or adult rats ( MΩ) (Wang et al., 2000), the input resistance of these type II cells (532 MWΩ to 8.46 GΩ) can be considered extremely high. Cells with such a high input resistance were also defi ned by others and named class 1 cells (Ambrogini et al., 2004a). Moreover, class 1 cells do not respond to medial perforant path (mpp) stimulation, indicating that they do not receive input from this brain region. In addition, no spontaneous activity could be recorded in these cells. Cells that do respond to mpp stimulation are named class 2 cells, which can in turn be subdivided in four subclasses, based on their different responses to the stimulation Maturation of the newly formed cells After becoming postmitotic, newly formed cells start to express the neuronal marker NeuN and the calcium-binding protein calretinin. In this stage, cells are named type 4 cells (Kempermann et al., 2004). During further maturation, cells switch from expressing calretinin to calbindin. New neurons rapidly form axons towards the CA3 region. Fast Blue and FluoroRuby injections in the CA3 region at different time points after BrdU injection demonstrate that, in rats, newly generated neurons start to grow axons to the CA3 area between 4 and 10 days after their formation (Hastings and Gould, 1999; Stanfi eld and Trice, 1988). Analysis of newly formed neurons that had retrovirally been labeled with GFP during cell division revealed that 4-week old neurons still have smaller soma than 4-month old granule neurons. Moreover, they have a shorter total dendritic length, a lower number 16

10 General introduction of branch points and a lower spine density (Van Praag et al., 2002). It takes two to four weeks before newly formed neurons are functionally indistinguishable from other neurons (Van Praag et al., 2002). Four-week old neurons are immunoreactive for synaptophysin and have dendritic spines, indicating that they receive synaptic input (Markakis and Gage, 1999; Van Praag et al., 2002). In addition, newly generated neurons display spontaneous postsynaptic currents, with a fast onset (less than 1 ms) and a slower exponential decay (greater than 5 ms), which is typical of postsynaptic response to fast neurotransmitters, such as GABA. Furthermore, new neurons receive input from the perforant path and they show mostly similar electrophysiological properties compared to mature dentate cells, indicating that newly generated neurons are functionally similar to mature cells (Van Praag et al., 2002) Fluctuations in newly formed cell number The estimated cell cycle time of proliferating hippocampal progenitors is h for rats and h for mice. The length of the S-phase, during which BrdU can be incorporated into the DNA, is estimated to be 9-10 h for rats and 6-9 h for mice (Cameron and McKay, 2001; Hayes and Nowakowski, 2002). BrdU is thought to label cells within 2 h after injection. After a single injection with BrdU, the number of labeled cells roughly doubles between 2 h and 24 h (Cameron and McKay, 2001; Dayer et al., 2003; Palmer et al., 2000). In rats, the BrdU-positive cell number continues to increase at least until day 7 after the initial labeling (Palmer et al., 2000), or even until day 10 (Hastings and Gould, 1999). In mice, the BrdU-positive cell number rises until day 3 (Jessberger and Kempermann, 2003; Kempermann et al., 2004; Kronenberg et al., 2003; Steiner et al., 2004). After the peak in the number of newly generated cells has been reached, the amount of labeled cells strongly decreases. This decrease is thought to be caused by apoptosis of the newly formed cells or by label dilution resulting from ongoing proliferation (Prickaerts et al., 2004). The latter explanation is less likely, because between 2 h and 4 days after labeling with BrdU, the BrdU-positive cell population loses expression of the proliferation marker Ki-67, indicating that the cells exit the cell cycle (Dayer et al., 2003). In addition, the neurogenic regions in the brain has been shown to contain high numbers of apoptotic cells (Biebl et al., 2000). Between two and four weeks after cells have been generated, the number of BrdU-positive cells does not decrease any further and remains stable for at least months (Cameron and McKay, 2001; Dayer et al., 2003; Hastings and Gould, 1999; Kempermann et al., 2003; Kempermann et al., 2004; Palmer et al., 2000). The net number of newly generated cells in the DG per day is estimated to be 9000 in 10-week old rats (Cameron and McKay, 2001), which is approximately 1% of the total granule cell population. The newly formed cell number rapidly diminishes with age (see section 4.2). The rate of hippocampal neurogenesis shows a large variation between species. The macaque, for instance, only forms 200 new hippocampal cells per day, which represents only 0.004% of the granule cell layer (Kornack and Rakic, 1999). 17

11 Chapter 1 The fact that many of the newly formed cells die soon after they have been generated, implies that the timing of BrdU-injections with respect to the sacrifice of the animals crucially influences the interpretation of the data. Injections that are applied hours to a few days before termination will provide information on the proliferation of undifferentiated precursor cells. However, if animals are injected with BrdU one week or more in rats or three days or more in mice before they are sacrificed, BrdU staining will give an indication about the survival of newly generated cells and allow double labelings to study the phenotype of the BrdU-positive cells. Therefore, strategic injections with BrdU in combination with immunocytochemical stainings for proliferation markers, such as Ki-67, and immature neuron markers, such as DCX, will result in a relatively complete picture of the entire neurogenic process in one individual animal. 4. Regulation of hippocampal neurogenesis 4.1. The neurogenic microenvironment Adult hippocampal neurogenesis is thought to take place in an exclusive neurogenic niche. This idea is supported by the observation that transplantation of stem cells from the hippocampus or from the spinal cord into the hippocampus gives rise to new neurons, whereas transplantation to different parts of the CNS, such as the spinal cord, does not result in neuronal differentiation (Shihabuddin et al., 2000). A possibly important factor for the creation of a permissive microenvironment for neurogenesis is the vasculature. Proliferating clusters in the SGZ tend to be located around small capillaries. This close proximity to the vasculature appears to be exclusive for the DG, because proliferating precursors in other areas of the hippocampus do not show this close association with a vascular niche (Palmer et al., 2000; Palmer, 2002). There is also in vitro evidence for a role of blood vessels in promoting neurogenesis. Coculturing of NSC s with endothelial cells, but not with vascular smooth muscle cells, has been reported to promote NSC proliferation (Shen et al., 2004). In addition, the presence of endothelial cells delays differentiation of the NSC s and stimulates the formation of neurons. Since this effect can be accomplished without direct cell-cell contact between NSC s and endothelial cells, endothelial cells probably produce soluble factors that promote neurogenesis. A possible candidate is the vascular endothelial growth factor vegf, which also has been shown to promote cell proliferation (Cao et al., 2004; Schanzer et al., 2004; Wurmser et al., 2004). Next to the vasculature, hippocampal astrocytes are also likely to play a role in neuronal fate instruction of hippocampal precursors (Horner and Palmer, 2003; Song et al., 2002; Svendsen, 2002). When NSC s are differentiated in the presence of astrocytes derived from the adult hippocampus, the number of neurons increases tenfold (Song et al., 2002). Astrocytes from the adult hippocampus are about twice as effective as astrocytes that are obtained from the neonatal hippocampus in promoting neurogenesis. Astrocytes 18

12 General introduction from the adult spinal cord do not stimulate neurogenesis (Song et al., 2002), indicating that adult hippocampal astrocytes provides specifi c cues that promote neurogenesis Internal and external factors that regulate hippocampal neurogenesis There is a large variety of factors that have been shown to influence hippocampal neurogenesis. Age is perhaps the most important variable to affect neurogenesis. Hippocampal cell proliferation occurs at high rates in very young rats, but as the animals reach the youngadult stage, proliferation has reduced with approximately 75% and it further declines to hardly detectable levels in aged animals (Heine et al., 2004a; Kempermann et al., 1998; Kuhn et al., 1996). Stress and glucocorticoids have also repeatedly been reported to decrease hippocampal neurogenesis. Injections with corticosterone, for instance, have a negative effect on hippocampal cell proliferation, whereas adrenalectomy promotes the generation of new cells (Cameron and Gould, 1994). Also, acute psychosocial stress (Gould et al., 1997) or chronic unpredictable stress have been shown to reduce progenitor proliferation (Heine et al., 2004b) and survival (Westenbroek et al., 2004) of newly formed cells in male rats. A similar chronic stress protocol in female rats, however, caused an increased survival of newly generated neurons (Westenbroek et al., 2004). Besides corticosterone, other hormones can also affect hippocampal neurogenesis. Estrogen, for instance, has complicated effects on hippocampal cell proliferation. Administration of a high dose of estradiol in female meadow voles initially increases cell production, but reduces it again after 48 hours (Ormerod et al., 2003). In female rats, progenitor proliferation fluctuates throughout the estrus cycle, with highest levels during proestrus, but net neurogenesis is not different from males (Tanapat et al., 1999; Tanapat et al., 2005). Several neurotransmitter systems are also potent inhibitors or activators of hippocampal neurogenesis. Glutamatergic signaling via the NMDA receptor, for instance, has been reported to have a negative effect on progenitor proliferation (Cameron et al., 1995). Reduction of NMDA receptor activation by pharmacological blockage of the NMDA receptor or by damaging the major excitatory input into the hippocampus, the entorhinal cortex, stimulates cell proliferation and the formation of new neurons (Cameron et al., 1995; Nacher et al., 2001b). Another neurotransmitter that has been shown to have an impact on neurogenesis is serotonin or 5-HT. Pharmacological inhibition of serotonin synthesis or selective lesions of the serotonergic neurons strongly reduce hippocampal cell proliferation (Brezun and Daszuta, 1999). In addition, antagonists for the 5-HT1A and 5-HT2A receptor decrease the number of dividing cells (Banasr et al., 2001; Radley and Jacobs, 2002). 5-HT1A receptor agonist treatment, on the other hand or stimulation of serotonergic neurotransmission by chronic administration of antidepressants promote the formation of new cells (Banasr et al., 2001; Malberg et al., 2000). Hippocampal neurogenesis is also positively influenced by a variety of growth fac- 19

13 Chapter 1 tors and neurotrophic factors. Peripheral (Aberg et al., 2000) or central (Lichtenwalner et al., 2001) administration of insulin-like growth factor-1 (IGF-1), central infusion of basic fi broblast growth factor (bfgf) or epidermal growth factor (EGF) (Jin et al., 2003a) or overexpression of vascular endothelial growth factor (vegf) (Cao et al., 2004) activate progenitor proliferation. VEGF also has survival-promoting effects on newly formed cells (Schanzer et al., 2004). Moreover, BDNF has also been shown to have positive effects on the survival of newly formed neurons and the differentiation towards the neuronal phenotype (Sairanen et al., 2005; Scharfman et al., 2005). Furthermore, pathological conditions can affect adult hippocampal neurogenesis. Global and focal cerebral ischemia (Kee et al., 2001; Liu et al., 1998a; Tureyen et al., 2004; Zhu et al., 2004a), excitotoxic and mechanic lesions of the granule cell layer (Gould and Tanapat, 1997), or epileptic seizures (Cha et al., 2004; Hellsten et al., 2002; Parent et al., 1997; Sankar et al., 2000) all result in increased levels of hippocampal neurogenesis. Also in humans, there appears to be a link between brain pathology and neurogenesis, because the hippocampi of Alzheimer patients were reported to contain more cells that express markers for immature neurons than age-matched healthy controls (Jin et al., 2004). In animal models of ischemia, newly formed cells have been shown to migrate to the damaged area where some of them differentiate into neurons and may contribute to repair (Arvidsson et al., 2001; Jin et al., 2003b; Nakatomi et al., 2002) Effects of physical exercise on hippocampal neurogenesis Part of this thesis will be focused on the relation between running wheel activity and hippocampal neurogenesis. Physical activity has repeatedly been shown to cause a robust enhancement of hippocampal cell proliferation and the number of newly formed neurons. Mice that are housed with a running wheel show a large increase in the proliferation of hippocampal progenitors and the production of new granule neurons (Van Praag et al., 1999a; Van Praag et al., 1999b). Also in rats, beneficial effects of running on hippocampal neurogenesis have been demonstrated. Female spontaneous hypertensive rats (SHR), like mice, show voluntary running behavior when their cage is equipped with a wheel, which increases the production of new neurons with approximately 500% (Persson et al., 2004). Also non-voluntary running in a treadmill can promote hippocampal neurogenesis in rats of different ages (Kim et al., 2004; Trejo et al., 2001), but only if the intensity level of the exercise is mild to moderate (Kim et al., 2003). Running-induced stimulation of cell proliferation is restricted to the early stages of granule cell development, when cells express nestin. Radial glia proliferation remains unaffected, but proliferation of type 2A and type 2B cells (Fig. 1) is increased. Mitotic cells which have already lost nestin expression remain unaffected by exercise (Kronenberg et al., 2003). Exercise only affects hippocampal neurogenesis and does not influence neuron formation in the other neurogenic region of the brain, the olfactory bulb (Brown et al., 2003a). 20

14 General introduction 5. Neurogenesis and learning Despite the increasing number of studies that have been performed during the last decades to unravel the function of neurogenesis in the adult hippocampus, it is still unclear why new neurons are being generated in this brain structure. The fact that hippocampal neurogenesis is a phenomenon that can be observed in a large variety of species, supports the hypothesis that newly generated neurons have a distinct role in hippocampal functioning. Because of the well-known role of the hippocampal formation in learning and memory processes, it is tempting to speculate on a potential role for newly formed neurons in learning. There is increasing experimental evidence that there is a relation between learning and neurogenesis, though the data that are obtained are not equivocal. Here, a summary will be provided showing the variety of studies that have been performed to explore the role of hippocampal neurogenesis in learning Environmental impact on hippocampal neurogenesis The fi rst studies that indicated a role for newly formed hippocampal neurons in learning and memory were carried out in wild-living animals. In black-capped chickadees (Parus atricapillus) (Barnea and Nottebohm, 1994), for instance, neurogenesis appears to be related to seasonal variation in spatial memory processing. The number of new neurons in these birds shows a seasonal fluctuation, with most new neurons being produced during fall, when birds have to retrieve the spots where they have hidden food hours to days earlier. Interestingly, captive birds show a similar seasonal fluctuation of neurogenesis, though the total rate of new neuron production is only half of the free-living chickadees. In contrast to the fi ndings in the chickadees, no seasonal variations in hippocampal neurogenesis were observed in another food-storing species, the eastern grey squirrel (Sciurus carolinensis) (Lavenex et al., 2000), which suggests that fluctuations in neurogenesis across the year may be specific for birds. However, there is evidence that also in mammals, the complexity of the environment may be a determining factor for the rate of hippocampal neurogenesis. A comparison between four species of wild-living rodents shows that the species with the largest territory (yellow-necked and long-tailed wood mouse, Apodemus spp.) display the highest level of hippocampal neurogenesis (Amrein et al., 2004). Also in laboratory animals, enriched housing causes almost a doubling of the number of new neurons in young-adult mice and rats or even a fi vefold increase in aged mice, without influencing proliferation of undifferentiated precursor cells (Kempermann et al., 1997; Kempermann et al., 1998; Kempermann et al., 2002; Nilsson et al., 1999). Moreover, environmental enrichment results in a better performance in the Morris water maze (Kempermann et al., 2002; Nilsson et al., 1999). The positive effect of an enriched environment on neurogenesis is restricted to hippocampal neurogenesis. The number of newly generated neurons in the olfactory bulb does not increase by enriched housing (Brown et al., 2003a). 21

15 Chapter Effects of learning on neurogenesis There is also more direct evidence that there is a relation between learning and hippocampal neurogenesis. It has been shown that hippocampus-dependent learning can promote hippocampal neurogenesis in adult rats (Gould et al., 1999a). Training of rats in the trace eyeblink conditioning task (TEC) or the Morris water maze (MWM), one week after an injection with BrdU, has been shown to result in a doubling of the number of newly generated neurons. The hippocampus-independent versions of TEC and MWM, delay eyeblink conditioning and cued water maze learning, respectively, did not alter hippocampal neurogenesis. Follow-up studies showed that the increased number of new neurons that are formed during TEC remain for months after learning, a time point when the hippocampus is not required anymore for the retrieval of trace memories (Leuner et al., 2003). Using another protocol, in which rats were trained in the MWM directly after BrdU injection and sacrificed ten days after the last training session, Ambrogini et al. (2000) also showed positive effects of spatial learning on neurogenesis, although those effects were restricted to the infrapyramidal blade of the dorsal granule cell layer. The same authors reported that training of rats in the MWM 8-10 days after BrdU injection caused a significant decrease in hippocampal neurogenesis (Ambrogini et al., 2004b). In another study in rats (Snyder et al., 2005), in which BrdU was injected one week before the start of training, and in mice (Van Praag et al., 1999b), in which BrdU was injected before and during MWM training, no learning-induced alterations in the number of newly generated neurons were observed. Not only cell survival can be influenced by learning, it has also been reported that hippocampal progenitor proliferation may increase during spatial learning in rats (Lemaire et al., 2000), though this phenomenon was not observed by others (Gould et al., 1999a; Van Praag et al., 1999b). There is also evidence for complex fluctuations in cell production throughout the learning process (Dobrossy et al., 2003). Cell proliferation has been shown to be stable during the early phase of Morris water maze learning, but to increase during the asymptotic phase of the learning curve. At the same time, cells that have been generated during the early phase of learning die during the later stages of the learning process. Therefore, the net level of cell production remains unaltered during learning. Moreover, there are data that indicate a positive correlation between progenitor proliferation and spatial learning capacity in aged rats (Drapeau et al., 2003), but this is contradicted by others (Bizon et al., 2004; Merrill et al., 2003). Associative learning tasks, such as fear conditioning and two-way active avoidance learning, were shown to reduce hippocampal cell proliferation (Malberg and Duman, 2003; Pham et al., 2005), whereas another associative task, TEC, had no effect on cell proliferation (Gould et al., 1999a). A summary of the studies that tested the effects of learning on hippocampal neurogenesis is provided in Table 1. 22

16 General introduction 5.3. Effects of reduced neurogenesis on learning A different approach to investigate the relation between hippocampal neurogenesis and learning is to block hippocampal cell proliferation and to look at the behavioral consequences. Two weeks of treatment with the non-specifi c mitosis-inhibitor methylazoxymethanol acetate (MAM), which reduces neurogenesis with approximately 80%, signifi - cantly impaired TEC and trace fear conditioning in rats (Shors et al., 2001; Shors et al., 2002). Partial reduction of neurogenesis is not detrimental for all types of hippocampusdependent learning. MAM treatment or brain irradiation, which also dramatically reduces hippocampal neurogenesis (Monje et al., 2002), had no effect on MWM learning (Madsen et al., 2003; Shors et al., 2002). Moreover, contextual fear conditioning, another hippocampus-dependent learning task, remained unchanged after injections with MAM (Shors et al., 2002). Although the acquisition of many hippocampus-dependent tasks is not impaired by inhibition of neurogenesis, recent studies suggest that neurogenesis may be crucial for the consolidation of memories. Almost total ablation of hippocampal neurogenesis impaired long-term memory retention in the Morris water maze (Rola et al., 2004; Snyder et al., 2005) and it prevented the environmental enrichment-induced improvement in longterm recognition memory (Bruel-Jungerman et al., 2005) Computational models of neurogenesis: neurogenesis and memory clearance Computational models have been used to predict the role of hippocampal neurogenesis in learning and memory. In a simple model of a three-layered network without backward or lateral signaling, in which new cells are randomly inserted into the middle layer and old cells are randomly deleted, removal of old memories is strongly accelerated. Moreover, acquisition of new memories is accelerated in the model containing neurogenesis, especially with increasing network activity (Chambers et al., 2004; Deisseroth et al., 2004). There is also in vivo evidence for al role for neurogenesis in memory clearance (Feng et al., 2001). Presenilin-1 conditional knockout mice do not show an increase in hippocampal neurogenesis upon cage enrichment. When these transgenic mice were trained in a contextual fear conditioning paradigm and subsequently housed in enriched conditions for two weeks, retention was signifi cantly better than in control mice in which neurogenesis was increased during the enrichment procedure. Without enrichment, both mouse lines performed similarly in the retention test. These data suggest that the increase in neurogenesis in the wildtype mice after enrichment removed old memories from the hippocampus. 23

17 Chapter 1 Table 1: Overview of studies exploring the eff ects of learning on hippocampal neurogenesis. Abbreviations: ASA: Active shock avoidance, CFC: Contextual Fear Conditioning, DEC: Delayed Eyeblink Conditioning, MWM: Morris Water Maze, TEC: Trace eyeblink conditioning. Publication Species, strain Age/weight Learning task Protocol Effect on BrdUpositive cell number Gould et al. (1999a) g MWM/ TEC BrdU (200 mg/kg): day -7 Training: between day 1 and 8 Sacrifice 24 h after training No change Gould et al. (1999a) g MWM/ TEC BrdU (200 mg/kg): day -7 Training: between day 1 and 8 Sacrifice 7 d after training Increase Gould et al. (1999a) g Cued MWM/ DEC BrdU (200 mg/kg): day -7 Training: between day 1 and 8 Sacrifice 7 d after training No change Van Praag et al. (1999b) Van Praag et al. (1999b) Female mouse, C57Bl/6 Female mouse, C57Bl/6 3 months MWM BrdU (50 mg/kg): day 1 to 12 Training: day 1 to 23 Reversal training: day 24 to 30 Sacrifice: day 40 3 months MWM BrdU (50 mg/kg): day 1 to 12 Training: day 1 to 12 Sacrifice: day 13 No change No change Ambrogini et al. (2000) 2 months MWM BrdU (50 mg/kg): day -3 to -1 Training: day 1 to 5 Sacrifice: day 15 Increase (infrapyramidal bade) Lemaire et al. (2000) 4 months MWM BrdU (unknown conc.): day 3 to 5 (before first trial) Training: day 1 to 5 Sacrifice: day 6 Increase 24

18 General introduction Table 1, continued Publication Species, strain Dobrossy et al. (2003) Dobrossy et al. (2003) Dobrossy et al. (2003) Malberg and Duman (2003) Ambrogini et al. (2004b) Snyder et al. (2005) Long Evans Pham et al. (2005) Age/weight Learning task Protocol Effect on BrdUpositive cell number 2 months, 200 g MWM BrdU (50 mg/kg): day 1 to 8 (before first trial) Training: day 1 to 8 Sacrifice: day 9 No change 2 months, 200 g MWM BrdU (50 mg/kg): day 5 to 8 (before first trial) Training: day 1 to 8 Sacrifice: day 9 Increase 2 months, 200 g MWM BrdU (50 mg/kg): day 1 to 4 (before first trial) Training: day 1 to 8 Sacrifice: day 9 Decrease g ASA BrdU (100 mg/kg): directly after training Training: 30 trials on 1 day Sacrifice: 2 h after BrdU Decrease 5 months MWM BrdU (50 mg/kg): day -10 to -8 Training: day 11 to 15 Sacrifice: day 18 Decrease ~2 months MWM BrdU (200 mg/kg): day -7 Training: day 1 to 6 Sacrifice: 1, 2 or 4 w after training No change 3 months CFC BrdU (200 mg/kg): immediately after CS exposure Training: pre-exposure to CS on day 1, exposure to CS + shock on day 2 Sacrifice: 2 h after BrdU Decrease 25

19 Chapter 1 6. Outline of this thesis The fi rst part of this thesis describes various factors that may influence hippocampal neurogenesis. In chapter 2, we investigated baseline fluctuations in hippocampal cell proliferation in mice. We assessed whether the cell cycle of hippocampal progenitors follows a circadian rhythm. Furthermore, this chapter describes the effects of acute sleep deprivation during the resting phase of the animals on the number of proliferating progenitors. Third, we studied if physical exercise, which mainly takes place during the active dark phase of the mice, induces a synchronization of the proliferating cell population. In chapter 3, the effects of exercise on hippocampal cell proliferation were investigated in more detail. In this chapter, we were interested in the temporal dynamics of the exercise-induced increase in neurogenesis. Are these effects acute or is long-term exercise required? And how long do the effects on hippocampal cell proliferation persist after removal of the running wheel from the cage? Chapter 4 describes an experiment in which we investigated the role of the medial septum in hippocampal neurogenesis. The medial septum forms one of the main input pathways to the hippocampus and it is an important brain structure for learning and memory. In addition, it is known that during physical activity, the septohippocampal connection is strengthened. Therefore, the medial septum can be considered as a possible mediator in the effects of learning or exercise on hippocampal neurogenesis. The studies described in the next three chapters investigate the potential role of hippocampal neurogenesis in learning and memory processes. Chapter 5 describes an experiment in which rats were exposed to either one or four days of active shock avoidance learning. These data provide insight into the effects of an associative learning task, which is hippocampus-independent, but which has been shown to activate cells in the dentate gyrus, on hippocampal cell proliferation and survival of newly formed cells. Chapter 6 explores the effects of a spatial learning paradigm, Morris water maze learning, on different aspects of hippocampal neurogenesis and plasticity. The study was performed with two frequently used strains, Wistar and. Chapter 7 also describes experiments related to spatial learning and neurogenesis. Using the Y-maze as a learning task, we investigated whether an increase in the number of newly formed neurons would increase the rate of acquisition in a spatial learning task. Neurogenesis was enhanced by housing animals with a running wheel for 14 days. Secondly, we tested the hypothesis that enhanced neurogenesis following acquisition results in the clearance of information from the hippocampus. Third, we explored potential changes in hippocampal neurogenesis during memory retrieval, when the hippocampal memory trace is reactivated. 26

20 General introduction 27

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