HIPPOCAMPUS 16:208–215 (2006) Functional Maturation of Adult-Generated Granule Cells Linda S. Overstreet-Wadiche* and Gary L. Westbrook ABSTRACT: The excitability and connectivity of adult-generated granule cells dictate to what extent newborn neurons participate in the hippocampal network. These functional parameters evolve as newborn cells mature and interact with the existing circuit. The progression of granule cell maturation during neonatal development appears to be reiterated in the adult, but with some caveats. New approaches to identify and track newborn neurons are revealing the timing of this process, as well as its sensitivity to activity-dependent regulation. V 2006 Wiley-Liss, Inc. C KEY WORDS: neurogenesis; hippocampus; dentate gyrus; synaptic INTRODUCTION A central question in stem cell biology is understanding how stem cells and their progeny interact with their environment. Adult neurogenesis in the dentate gyrus (DG) provides one model system to investigate this issue. To participate in the hippocampal circuit, adult-generated cells must acquire neuronal functions, such as membrane excitability, and interact with mature neurons to establish synaptic connectivity. Studies of hippocampal progenitors in vitro demonstrate that these processes are inﬂuenced by environmental factors (Song et al., 2002a,b). Just as neuronal properties progressively mature during neonatal development, newborn granule cells in adults progress through sequential stages of functional maturation. Recent work suggests that this progression is a dynamic process that can be modulated by differing environmental conditions. In this review, we summarize current understanding of the functional maturation of adult-generated granule cells, with an emphasis on new techniques that allow identiﬁcation of adult-generated cells at discrete postmitotic ages. PROPERTIES OF IMMATURE GRANULE CELLS DURING NEONATAL DEVELOPMENT Insights into the maturation of adult-generated granule cells can be gleaned from studying granule cell development during the neonatal period. In rodents, granule cell neurogenesis begins between embryonic day 10 and 14, in a narrow area of neuroepithelium adjacent to the ﬁmbria (Angevine, 1965; Schlessinger, 1975; the primary dentate matrix, Altman and Bayer, 1990). Postmitotic immature granule cells and preVollum Institute, L474, Oregon Health & Science University, Portland, Oregon Grant sponsor: NIH Grant number: NS26494. *Correspondence to: Linda Overstreet-Wadiche, Vollum Institute, L474, Oregon Health & Science University, Portland, OR 97239. E-mail: firstname.lastname@example.org Accepted for publication 1 November 2005 DOI 10.1002/hipo.20152 Published online 12 January 2006 in Wiley InterScience (www.interscience. wiley.com). C V 2006 cursors then migrate into the DG. Subsequent generations of granule cells are generated in the secondary matrix and, like adult-generated cells, in the tertiary matrix of the subgranular zone. The granule cell layer (GCL) forms in an outside-in pattern, such that the oldest granule cells are located near the molecular layer and newly born cells are near the subgranular zone. The peak of granule cell neurogenesis occurs in the secondary and tertiary matrix at the end of the ﬁrst postnatal week and declines over subsequent weeks (Schlessinger et al., 1975). The prolonged window of granule cell neurogenesis means that granule cells at different developmental stages and postmitotic ages coexist within the GCL. Trommer and colleagues took advantage of this intermingling to compare the physiology of individual granule cells at various developmental stages (Liu et al., 1996; Liu et al., 1998; Liu et al., 2000; Ye et al., 2000; Ye et al., 2005). Patch-clamp recordings during the ﬁrst postnatal month revealed a range of input resistances (IR, a measure of the number of ion channels open at the test membrane potential) and resting membrane potentials (RMP, a function of the nongated ion channels and the concentration gradients of their permeate ions) in neighboring granule cells. IR was inversely correlated with dendrite length, a morphological measure of granule cell maturity (Fig. 1a). Thus, IR could be used, even in the absence of morphological data, as an indicator of granule cell maturity. IR was also correlated with the RMP. Indeed, high IR and depolarized RMP are hallmarks of immature neurons in many embryonic preparations. Unlike most regions where the IR and RMP shift to mature values in parallel with the age of the animal, some dentate granule cells with very immature properties were present at the end of the ﬁrst postnatal month, suggesting continued neurogenesis. Furthermore, the most mature granule cells at the end of the ﬁrst postnatal week had the same properties as the most mature cells at the end of the ﬁrst postnatal month, suggesting that IR and RMP reached their mature values within weeks of ﬁnal cell division. Synaptic input to granule cells likewise changes dramatically during neonatal development. The major afferent input, the perforant path, arises from pyramidal cells in the entorhinal cortex (EC). The axons of the medial and lateral entorhinal areas are strictly segregated in the middle and outer molecular layer, respectively. These inputs originate at the middle of the ﬁrst postnatal week (Fricke and Cowan, 1977). At WILEY-LISS, INC. FUNCTIONAL MATURATION OF ADULT-GENERATED GRANULE CELLS 209 FIGURE 1. Immature granule cells identiﬁed by IR and morphology. a: Examples of biocytin-ﬁlled granule cells with immature, intermediate, and mature IR and morphology in juvenile rats. Arrows indicate labeled axons. Scale bars, 20 mm. Modiﬁed with permission from Elsevier: Brain Research 856:202–212, 2000. Characteristic synaptic currents and membrane potential responses (insets) are shown below. Cells with high IR had prominent slow GABAA receptor-mediated currents (asterisk) and small prolonged action potentials (arrowhead). Cells with lower IR and more mature morphology had larger glutamatergic synaptic currents and fast action potentials. Scale bars, 50 pA, 20 ms; 40 mV, 20 ms (insets). Modiﬁed with permission from The American Physiological Society: Journal of Neurophysiology 76:1074–1088. b: Granule cells at similar stages of maturation were found in young adult rats. Examples of biocytin-ﬁlled cells (green, top panels) and volt- age responses to current injections (middle panels) and synaptic responses to stimulation in the molecular layer (lower panels). Granule cells with the most immature morphology (left panels) either did not ﬁre action potentials or had only small action potentials. These cells lacked synaptic input or had only GABAA receptor-mediated responses (blocked by bicuculline, BMI). Cells with larger dendritic arbors and more mature membrane properties had progressively greater glutamatergic synaptic input, blocked by the AMPA receptor antagonist CNQX, and the NMDA receptor antagonist AP5 (middle and left panels). Scale bars 20 mm (top), 50 ms, 20 mV (middle), and 50 ms, 20 pA (bottom). Modiﬁed with permission from Elsevier: Brain Research 1017:21–31, 2004. [Color ﬁgure can be viewed in the online issue, which is available at www.interscience.wiley.com.] 210 OVERSTREET-WADICHE AND WESTBROOK 2004). Thus, immature granule cells have a lower threshold for induction of LTP and LTD. Manipulations that enhance neurogenesis, such as exercise, are correlated with greater LTP of population responses (Van Praag et al., 1999; Farmer et al., 2004). Enhanced plasticity in granule cells with immature properties strengthens the idea that enhanced plasticity is causally related to greater numbers of immature granule cells. Reduced numbers of newborn granule cells in irradiated animals was also associated with decreased LTP (Snyder et al., 2001). this time, immature granule cells are already surrounded by a well-developed plexus of GABAergic axons (Lubbers and Frotscher, 1988). Between postnatal day 5 and 12, Ye et al., (2000) found that granule cells could be segregated based on their response to stimulation in the middle molecular layer. Granule cells with the most immature membrane properties had either no synaptic response, exclusively GABAergic synaptic input, or small NMDA receptor-mediated responses. Granule cells with more mature IR and RMP had both AMPA and NMDA-receptor mediated EPSCs. This sequence of synaptogenesis is similar to CA1 pyramidal cells, where GABAergic input appeared before glutamatergic responses that were mediated sequentially by NMDA and then AMPA receptors (Tyzio et al., 1999). However, until postnatal day 4, granule cells had exclusively GABAergic synapses (Hollrigel and Soltesz, 1997). The subsequent appearance of glutamatergic input from the perforant path parallels dendrite elongation and spine formation in the molecular layer at the end of the ﬁrst postnatal week (Jones et al., 2003). ADULT-GENERATED GRANULE CELLS LABELED WITH RETROVIRAL VECTORS Granule cells with immature membrane properties clearly have distinct physiological behavior compared to granule cells with mature properties. However, identiﬁcation of adult-generated granule cells exclusively by their immature properties has limitations. For example, it is difﬁcult to assess maturity based on IR and RMP because reported values vary widely, presumably, reﬂecting both developmental variation and technical differences such as seal resistance. This approach also cannot determine whether adult-generated granule cells retain distinct properties once they have completely matured, nor can it provide information about the postmitotic age of immature cells. Labeling dividing cells with retroviral vectors that express green ﬂuorescent protein (GFP; Lewis and Emerman, 1994) is one way to address these issues. Once incorporated into progenitors during mitosis, subsequent newborn cells express GFP and can be visualized in living preparations. van Praag et al. (2002) used this technique to prove that adult-generated granule cells can become integrated into the hippocampal circuit. Four to eight weeks after viral injections, labeled granule cells had similar membrane and synaptic properties as unlabeled mature cells (Fig. 2a). By 4 months, labeled adult-generated granule cells also achieved dendrite complexity and spine number comparable to mature granule cells. These results support the view that, given sufﬁcient time, adult-generated granule cells become indistinguishable from neonatal-generated cells. However, more subtle measures, such long term plasticity, have not yet been compared. Retroviral labeling in organotypic slice cultures revealed various phenotypes of stem cell progeny, including early and late stages of differentiating neurons, as well as GFAP- and nestin-expressing progenitors (Kamada et al., 2004). Studies using viral labeling to identify adult-generated granule cells after various time windows will certainly provide valuable information about their physiology and progression of maturation (Esposito et al., 2005). In addition, this approach can be used to genetically alter adult-generated cells to study the molecular mechanisms of their development (Tashiro et al., 2004; Ge et al., 2005). IMMATURE GRANULE CELLS IN THE ADULT DG Immature granule cells in adult animals can also be identiﬁed by similar membrane properties and morphology. Ambrogini et al. (2004) classiﬁed granule cells in young adults based on their responses to perforant path stimulation (Fig. 1b). Similar to neonates, granule cells with the most immature membrane properties had either no synaptic input or exclusively GABAergic synaptic input. Granule cells with progressively more mature membrane properties and morphology also had glutamatergic synaptic responses. Immature granule cells were located exclusively near the border of the subgranular zone, whereas more mature cells were scattered throughout the GCL. Wang et al. (2000) also reported morphological and physiological differences between granule cells located in the inner and outer GCL. Although they had mature IR, granule cells in the inner GCL had more compact dendritic arbors and fewer primary branches compared to those in the outer CGL. Both populations had perforant path input, but interestingly, plasticity of synaptic transmission differed between the two groups. Granule cells in the inner GCL had greater paired-pulse facilitation, a measure of release probability. They also displayed long-term potentiation (LTP) whereas LTP in granule cells of the outer-layer only occurred when GABAergic inhibition was blocked (Wang et al., 2000). Schmidt-Hieber et al. (2004) showed that granule cells identiﬁed by their immature membrane properties and morphology had a lower threshold for LTP induced by u-burst stimulation. This may involve Ca2þ spikes mediated by T-type channels (Ambrogini et al., 2004; Schmidt-Hieber et al., 2004), that could promote LTP by enhancing postsynaptic Ca2þ entry and relief of the Mg2þ block of NMDA receptors. Pairing EPSPs with subthreshold Ca2þ spikes also resulted in long-term depression (LTD) in immature, but not mature, granule cells (Bischofberger et al., PROPERTIES OF NESTIN-EGFP PROGENITORS Adult-generated cells can also be identiﬁed using ﬂuorescent markers expressed under transcriptional control of developmen- FUNCTIONAL MATURATION OF ADULT-GENERATED GRANULE CELLS 211 currents and in some cases, voltage gated Naþ currents. Some Type II cells also ﬁred action potentials and displayed spontaneous GABAergic synaptic currents (Tozuka et al., 2005; Wang et al., 2005). Type II cells likely constitute a group of highly proliferative ‘‘transient amplifying cells’’ that can further be divided based upon sequential marker expression (Kronenberg et al., 2003). PROPERTIES OF POMC-EGFP NEWBORN GRANULE CELLS It has been proposed that an important milestone of granule cell development is the transient postmitotic stage (stage 5; Kemperman et al., 2004). Newborn granule cells in which the proopiomelanocortin (POMC) promoter drives EGFP appear to be at this critical junction. POMC, a pro-hormone that regulates feeding and energy homeostasis, is normally expressed in the pituitary and hypothalamus. Transgenic POMC-EGFP mice had EGFP expression colocalized with POMC peptides in those brain regions (Young et al., 1998). However, several independent lines of mice also had expression in the DG that was not correlated with POMC mRNA or protein (Overstreet et al., 2004b). Dentate expression required cryptic promoter sequences distinct from those required for expression in POMCproducing cells of the pituitary. Thus, POMC-EGFP in the DG appears unrelated to expression of the POMC gene product. However, several lines of evidence indicated POMC-EGFP was expressed by newborn granule cells. First, labeled cells had the morphology of newborn granule cells with small cell bodies located near the border of the hilus and granule cell layer. They had a primary dendrite that branched within the inner molecular layer but did not extend through the middle and outer molecular layers. They had basal neurites, and their axons projected through the hilus to stratum lucidum of CA3. They did not have mature mossy ﬁber boutons but rather their axons were studded with many small diameter varicosities. Second, EGFP-labeled cells had membrane properties consistent with immature neurons (Fig. 2). They had a high IR ($8 G X) and depolarized RMP. They ﬁred small and broad action potentials mediated by Naþ channels, characteristic of immature neurons. Third, they expressed markers of immature granule cells such as PSA-nCAM, but they lacked GFAP immunoreactivity. Fourth, they showed transient colabeling with BrdU. Finally, the number of EGFP-labeled cells declined with age and was enhanced by exercise, parallel to age- and exercise-induced changes in neurogenesis. The timing of POMC-EGFP expression can be compared to the expression of various developmentally-regulated proteins that are used to deﬁne stages of neural stem cell maturation (Fig. 3a). For example, pulse-chase experiments with a single injection of BrdU showed that dividing precursors express nestin and doublecortin (Brown et al., 2003; Fukuda et al., 2003). Doublecortin expression continues for weeks, overlapping with other immature markers such as PSA-nCAM and bIII tubulin. Conversely, NeuN colabeling was detected 10 days after BrdU incorporation, and gradually increased until >85% of BrdUþ FIGURE 2. Adult-generated granule cells identiﬁed with viral and genetic labeling. a: An example of an adult-generated granule cell expressing GFP (green) 4 weeks after injection of a retroviral vector. Four to eight weeks after labeling, adult-generated granule cells had dendrites that extended through the molecular layer and they received excitatory input from the perforant path. Current injection (Im, lower panel) revealed labeled granule cells had mature membrane properties, including low input resistance and large action potentials. Scale bars, 50 ms, 25 mV. Modiﬁed with permission from Macmillan Publishers Ltd: Nature 415:1030– 1034, 2002. b: In POMC–EGFP transgenic mice, EGFP is expressed in newborn granule cells (green) with immature properties. Compared to mature granule cells ﬁlled with biocytin (red) or 4week-old adult-generated granule cells (a), POMC–EGFP granule cells had short dendrites and small cell bodies. The arrow points to an EGFP-labeled cell ﬁlled with biocytin. Scale bar, 50 mm. POMC–EGFP cells had high IR and small prolonged action potentials (lower panel). GCL, granule cell layer; IML, inner molecular layer. Modiﬁed from The Society for Neuroscience: The Journal of Neuroscience 24:3251–3259, 2004. [Color ﬁgure can be viewed in the online issue, which is available at www.interscience. wiley.com.] tally-regulated promoters. This enables reliable, although transient, labeling of an entire population of adult-generated cells. GFP expressed by neural-speciﬁc regions of the nestin promoter is one such example (Yamaguchi et al., 2000). Interestingly, the morphology and membrane properties of nestin-GFP cells indicated at least two functional populations (Filippov et al., 2003; Fukuda et al., 2003; ). Type 1 cells were radial glial cells with stem-cell neurogenic potential (see also Seri et al., 2001). These cells expressed the glial marker GFAP and had astrocytic properties including low input resistance, a relatively linear currentvoltage relationship, A-type potassium currents, and a resting potential near the Kþ equilibrium potential. The second population of nestin-GFP cells lacked GFAP expression and astrocytic properties. Rather, these type II cells were smaller and had high input resistance, rapidly inactivating voltage gated Kþ 212 OVERSTREET-WADICHE AND WESTBROOK ture granule cells identiﬁed by IR and cell morphology (Liu et al., 2000; Ye et al., 2000; Ambrogini et al., 2004). POMC– EGFP cells had the same properties in adults and neonates, indicating that EGFP marks newborn granule cells at the same functional stage regardless of the age of the mouse. POMCEGFP expression also provides information about the timing of granule cell maturation. For example, the lack of excitatory input indicates it takes at least 2 weeks for the majority of adult-generated granule cells to receive input from the perforant path. This is consistent with the appearance of dendritic spines at 2–3 weeks after viral labeling (Zhao et al., 2004; Esposito et al., 2005). The initial appearance of GABAergic synapses on newborn granule cells is consistent with a putative trophic role for GABA in development (Owens and Kriegstein, 2002). During the ﬁrst postnatal week, granule cells have GABAergic synaptic input with slow kinetics (Hollrigel and Soltesz, 1997) and depolarized reversal potentials (Hollrigel et al., 1998). Similarly, in neonates and adults, synaptic currents in POMC–EGFP labeled granule cells had slow kinetics, presumably due to the absence of the a1 GABAA receptor subunit (OverstreetWadiche et al., 2005). Slow, depolarizing GABA-mediated synaptic responses may promote Ca2þ entry via voltage-gated Ca2þ channels. Consistent with this idea, the ClÀ reversal potential in newborn granule cells was $30 mV more positive than in neighboring mature cells (À45 mV compared to À75 mV; Overstreet et al., 2005), in the range of Ca2þ channel activation (Schmidt-Hieber et al., 2004). Because the action potential threshold in newborn cells was still more depolarized ($38 mV), GABA receptor activation usually did not drive ﬁring but rather shunted action potentials induced by current injection. In this sense, GABAergic responses were depolarizing but not necessarily excitatory (Staley and Mody, 1992). Although the measured RMP of newborn cells in whole-cell recording was also near the ClÀ reversal potential, such measurements are likely artiﬁcially low due to current leak through the pipette seal (Barry and Lynch, 1991). The appearance of spontaneous Ca2þ inﬂux blocked by GABAA receptor antagonists (unpublished observations) strongly suggests that newborn granule cells have depolarizing GABAergic synaptic responses that activate voltage-gated Ca2þ channels. FIGURE 3. POMC–EGFP is selective for newborn granule cells. a: The temporal expression pattern of developmentally regulated markers in adult-generated granule cells can be determined by colabeling with BrdU at multiple time intervals. Nestin and doublecortin (DCX) are expressed by neural progenitors and in the case of doublecortin, by newborn postmitotic neurons. The mature granule cell marker NeuN is subsequently expressed. NeuN and doublecortin data are from Brown et al., 2003; Nestin data is from Fukuda et al., 2003. The POMC–EGFP expression proﬁle indicates it is selective for newborn granule cells that are nearly 2 weeks postmitotic. Consistent with this timing, POMC–EGFP labeled cells did not express nestin (b), or high levels of NeuN (c). However, EGFP-labeled newborn granule cells expressed doublecortin (d). [Color ﬁgure can be viewed in the online issue, which is available at www.interscience. wiley.com.] cells expressed this marker for mature granule cells (Brown et al., 2003). The temporal pattern of POMC-EGFP expression reveals that it is selective for immature, but differentiated, neurons. In adults, peak colabeling between BrdU and EGFP occurred at 12 days and declined before 30 days (Overstreet et al., 2004a,b). The absence of BrdU colabeling at short (<3 days) and longer (30 days) intervals indicates that labeled cells are not dividing precursors nor mature granule cells. Consistent with this idea, POMC-EGFP granule cells did not express nestin nor signiﬁcant levels of NeuN, but they did express doublecortin (Fig. 3b–d). The uniform membrane properties of POMC–EGFP granule cells further indicate they constitute a relatively narrow developmental stage deﬁned by their functional characteristics. At this stage, newborn granule cells expressed glutamate receptors, but they lacked MPP synaptic input, likely because their short dendrites did not extend through the middle molecular layer. However, they were surrounded by a dense network of GABAergic boutons and they received GABAergic synaptic input (Overstreet et al., 2004b; Overstreet-Wadiche et al., 2005). Thus they are similar to the physiologically most imma- THE TIMING OF ADULT-GENERATED GRANULE CELL FUNCTIONAL MATURATION A working model for the progression of granule cell maturation can be generated by synthesizing the results described earlier with studies of BrdU labeling and marker expression (Fig. 4). Some interesting, and perhaps unexpected, developmental patterns emerge. For example, the IR of neural stem cells is initially very low (<100 MX), typical of astrocytes with high Kþ permeability. This rapidly changes as asymmetrical cell division generates much smaller transiently-amplifying cells followed by newborn granule cells with a > 10-fold higher IR. Over the next 2 months, the IR gradually approaches the value expected of FUNCTIONAL MATURATION OF ADULT-GENERATED GRANULE CELLS 213 FIGURE 4. Functional maturation of adult-generated granule cells. Stages of granule cell maturation based on functional studies. Information about the timing of each stage was provided by the complementary techniques of retroviral and genetic labeling. Labeling with developmentally regulated promoters (nestin-GFP, POMC– EGFP) provides timing information for an entire population of cells. This allows the average age of the population to be determined, but the precise age of any individual cell is unknown. Conversely, viral labeling places a precise upper age limit on individual cells, but fewer cells are labeled. The lack of age information for immature granule cells identiﬁed by their immature membrane properties is indicated by a (?). Asterisks indicate data for four- to eight-week-old granule cells from van Praag et al. (2002). The timing of granule cell maturation might be altered by conditions including exercise, seizures, and aging. Very recent studies with retroviral labeling corroborate this sequence of granule cell integration (Esposito et al., 2005; Ge et al., 2005). [Color ﬁgure can be viewed in the online issue, which is available at www.interscience.wiley.com.] mature granule cells ($300 MX). Proliferation and differentiation of several cell types is regulated by the expression of speciﬁc subtypes of Kþ channels (Kotecha and Schlichter, 1999; Cahalan et al., 2001; Chittajallu et al., 2002), but it is unknown whether similar mechanisms regulate dentate progenitors. The data also suggest that various aspects of neuronal function reach maturity at different times. For example, $2 week old granule cells that express POMC–EGFP have immature action potentials that rapidly mature as glutamatergic input arises over the next weeks (Ge et al., 2005). However, glutamatergic synapses on immature granule cells could exhibit enhanced synaptic plasticity for many subsequent weeks. Similarly, some newborn neurons have mature IR and RMP by 4 weeks, but spine growth and dendritic complexity increase for at least 8 weeks (van Praag et al., 2002). Although newly generated mossy ﬁber axons reach CA3 in the early weeks of granule cell genesis (Hastings and Gould, 1999; Overstreet et al., 2004b), nothing is known about the maturation of granule cell output. PLASTICITY OF GRANULE CELL FUNCTIONAL MATURATION The step-wise progression of granule cell development provides many points where maturation could be modulated. Indeed, studies using marker expression suggest that maturation is altered by conditions including exercise, seizures, and aging (e.g., Brandt et al., 2003; Kronenberg et al., 2003; Rao et al., 2005). POMC–EGFP expression is prolonged in adults compared to neonates, suggesting this functional stage is delayed in the adult environment that lacks the neonatal patterns of depolarizing network activity (Overstreet et al., 2004a). Pathological conditions can also alter the rate of granule cell maturation. After pilocarpine-induced seizures, POMC–EGFP labeled granule cells have elongated dendrites and they receive excitatory input (Bromberg et al., 2004). These results suggest that the functional integration, as well as proliferation (Deisseroth et al., 2004), of newborn granule cells is sensitive to activity in the 214 OVERSTREET-WADICHE AND WESTBROOK pocampus shows electrophysiological and morphological characteristics of astrocytes. Mol Cell Neurosci 23:373–382. Fricke R, Cowan WM. 1977. 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