壓力荷爾蒙與神經細胞之再生及死亡

行政院國家科學委員會專題研究計畫 成果報告

壓力荷爾蒙與神經細胞之再生及死亡(第 2 年)

研究成果報告(完整版)

計 畫 類 別 ： 個別型 計 畫 編 號 ： NSC 98-2314-B-004-001-MY2 執 行 期 間 ： 99 年 08 月 01 日至 100 年 10 月 31 日 執 行 單 位 ： 國立政治大學神經科學研究所 計 畫 主 持 人 ： 賴桂珍 報 告 附 件 ： 出席國際會議研究心得報告及發表論文 公 開 資 訊 ： 本計畫涉及專利或其他智慧財產權，2 年後可公開查詢

中 華 民 國 101 年 01 月 31 日

中 文 摘 要 ： 海馬迴是腦中負責學習與記憶的重要部位，也是許多神經疾 病的發病點，像是阿茲海莫症，失憶症，中風，癲癇，長期 壓力等等。如果能找到方法來修復腦部的病變，將可造福很 多人。在成人或成鼠的大腦裡有兩個地方可以持續的進行神 經細胞新生，海馬迴的粒細胞是其中一個。這個研究主要要 建立一個模範系統來研究在神經細胞死亡後如何可以處促進 其再生並恢復腦部功能。這個研究主要包括下列四部份: (1) 一個可以引起漸進特定細胞死亡的方式，(2)這些細胞的死亡 必需造成某種行為能力喪失(3)尋找可以次刺激神經細胞再生 及維持新細胞存活的物質(4)測試是否新細胞可以恢復腦部受 損功能。 移除腎上腺可以很專一的造成海馬迴粒細胞死亡而不影響腦 部任何其他區域，我將以此作為研究系統來建立神經細胞再 生並恢復腦部功能的模範系統，並借此更加了解神經細胞再 生並恢復腦部功能的機制。利用個體本身內生性’幹’細胞 來再生細胞將可免除手術不便，幹細胞來原的問題，以及異 體移植所產生的排斥。 中文關鍵詞： 海馬迴，神經新生

英 文 摘 要 ： The hippocampus is a brain region central to learning and memory and is a key target of many neurological diseases that have dramatic cognitive consequences, including Alzheimer’s and other forms of dementia, stroke, epilepsy, and chronic stress. Discovering methods that reverse damage would dramatically improve health for many people. Hippocampal granule cells are one of the two cell pools that produce new neurons continuously in adult mammalian brains. The proposed research uses a simple model system to study regeneration of lost neurons and restoration of

cognitive and physiological functions of the hippocampus. To achieve this goal, there are four necessary steps: 1. A model where gradual hippocampal neuron death can be induced； 2. A method for

characterizating the behavioural deficit associated with granule cell death； 3. Selection of substances and/or environmental stimulation that promotes

generation of new neurons； 4. Assays for determining if the new neurons are responsible for restoring the function of lost neurons. I will use adrenalectomy

surgery, removal of adrenal glands (ADX), to specifically eliminate granule cells in the hippocampus to study the regeneration of granule cells and restoration of functional brain circuitry. Corticosterone, an adrenal stress hormone, is

essential for the survival of granule cells. ADX leads to extensive granule cell death over a period of several weeks and gradually causes memory

deficits, especially in spatial tests. Many

substances are known to accelerate neurogenesis, but there are few data regarding the restoration of functional brain network after increased

neurogenesis. I will use a single factor or a

cocktail approach, consisting of several factors that promote neurogenesis (neurotransmitters, growth

factors, cell cycle factors) and treatments that enhance survival of granule cells (stimulating environment, corticosterone replacement) will be applied to ADX animals. The number and activity of granule cells will then be measured using

immunohistochemistry, electrophysiology and cognitive assays. This is the first model system for studying the regeneration of selectively and gradually lost neurons, regrowth of synaptic connectivity, and recovery of cognitive function. This work offers the promise to repair brain damage through neural circuit regeneration.

行政院國家科學委員會補助專題研究計畫

þ

_{□期中進度報告}

成果報告

壓力荷爾蒙與神經細胞之再生及死亡

計畫類別：þ個別型計畫 □整合型計畫

計畫編號：NSC

98-2314-B-004-001-MY2

執行期間：2009 年 8 月 1 日至 2011 年 10 月 31 日

執行機構及系所：國立政治大學神經科學所

計畫主持人：賴桂珍

共同主持人：

計畫參與人員：吳君逸，李思諭，連文瑜，林庭芳

成果報告類型(依經費核定清單規定繳交)：□精簡報告 þ完整報告

本計畫除繳交成果報告外，另須繳交以下出國心得報告：

□赴國外出差或研習心得報告

□赴大陸地區出差或研習心得報告

þ出席國際學術會議心得報告

□國際合作研究計畫國外研究報告

處理方式：

除列管計畫及下列情形者外，得立即公開查詢

þ涉及專利或其他智慧財產權，□一年□二年後可公開查詢

þ四年後可公開查詢

中 華 民 國 101 年 1 月 28 日

壓力荷爾蒙與神經細胞之再生及死亡

1. Objectives:

The hippocampus (HPC) is a key target of many neurological diseases, such as Alzheimer’s and other form of dementia, stroke, epilepsy, chronic stress which have dramatic cognitive consequences. The dentate gyrus(DG) subgranular zone of the hippocampus is the source of new born granule cells and is one of the 2 regions that continuously generate new neurons in adult mammalian brains. The proposed research aims to develop a simple mammalian model system for ablation and regeneration of specific neurons (granule cells) for studying the ability of regenerated neuron to restore cognitive and physiological functions of hippocampus. I have initiated part of the study when I was a post doctoral fellow at a laboratory specialized in hippocampal function and I currently expand this research to provide a model system for understanding the required conditions for replacing neural circuitry in the brain.

Overall Hypothesis: I propose that the endogenous pools of neurogenic stem/progenitor cells in the

hippocampus can be induced to replace lost neurons (granule cells) and form appropriate connections in such a way as to restore normal function.

II. Background and Significance: The capacity of the brain to exhibit plasticity when cells are lost due to injury or disease has been a core area for many decades in neuroscience research. Despite an impressive list of plastic processes, the progress on restoring neural circuits is strikingly limited. This reflects the extraordinary difficulty of the problem rather than shortcomings of the investigators. Nearly all of the work has involved one of two strategies: 1) acting on transmitter, modulator, or neurotrophin receptors, or 2) transplanting embryonic tissue or grafts derived from stem cells from a donor. The former strategy likely facilitates compensation in spared circuitry and there is no evidence for replacement of lost cells. The latter approach has been unimpressive in several ways: lack of long-term survival of grafted neurons, lack of evidence that grafted cells function as neurons, lack of evidence for integration of transplants into normal pre- and post-synaptic information processing positions in networks, and lack of availability of embryonic tissue (Lindvall & Hagell, 2002). Each of these is an important limitation. Even in instances of excellent graft survival with multiple transplant locations, there are clear persisting functional deficits (Helene et al., 2003; Shetty and Turner, 1996; Turner and Shetty, 2003).

Recently a new set of opportunities has opened up based upon the surprising discovery that in the adult brain there are two pools of cells that continuously generate new cells, including neurons. One of these pools of neurogenic stem cells is centered on the subventricular zone of the lateral ventricular wall and the other, the focus of this proposal, is located in the dentate subgranular zone of the HPC .

If this model is to be successfully developed four conditions must be satisfied, it must be possible to: (1) selectively eliminate the DG granule cells; (2) behavioral deficits associated with granule cell death; (3) Selection of substances and/or environmental stimulation that promotes generation of new neurons; (4)

demonstrate that recovery of deficits is due to regenerating the granule cells and connections. II-1. Selective elimination of hippocampal granule cells

Moderate level of the corticosterone (CORT), secreted by adrenal gland, is essential for hippocampal granule cell survival. Removal of adrenal glands (adrenalectomy, ADX) can lead to specific granule cell death in the hippocampus within a few weeks without significant effects to other regions of the brain (Sloviter et al., 1989, 1993a, 1993b; Sousa et al., 1997). Thus, we will use this model to selectively remove granule cells. After ADX, corticosterone levels, weight gain, and salt intake, which are all affected by ADX, will be assessed in each ADX-rat to confirm that the adrenal glands were successfully removed.

The lack of stimulation of the high-affinity glucocorticoid receptors leads to apoptotic cell death, only in granule cells. Thus, unlike with some other lesion methods there is a procedure available to evaluate side effects unrelated to granule cell death. Given the differences between abrupt vs. prolonged lesion processes, another attractive feature of ADX-induced granule cell death as a model, involves the relatively gradual loss of neurons, a feature more in line with neural degenerative diseases.

II-2. Deficits after hippocampal granule cell loss

Certain learning and memory processes are known to depend upon HPC circuit activity in rodents. Examples of tasks that show a large deficit after HPC damage in rats include the hidden platform version of the Morris water task (Morris et al 1982; Sutherland, et al., 1982), Pavlovian conditioned fear of context (Kim and Fanselow, 1992; Phillips and LeDoux, 1994; Sutherland and McDonald, 1990), and delayed recognition memory tasks involving visual or odor cues (Dudchenko et al. 2000; Prusky et al., 2004a). Even within these tasks it is possible to arrange variations in procedure that make the task more or less sensitive to HPC damage. Another issue concerns whether selective loss of granule cells produces behavioral deficits similar to those after complete (or nonselective) HPC damage. Reliable deficits after lesions largely confined to the DG have been reported in the Morris water task, radial arm maze,

Hebb-Williams maze, spatial pattern separation contextual fear conditioning, general activity levels, and exploration (Czurko et al., 1997; Gilbert et al., 2001; Jeltsch et al., 2001; Lee & Kesner 2004a & b;, 1983; Tandon et al., 1991; Xavier et al., 1999 ).

II-3. Hippocampal Neurogenesis

There are at least two brain regions in which neurogenesis in adult animals has been demonstrated, a forebrain subventricular zone (SVZ) and the HPC subgranular zone (SGZ) (Alvarez-Buylla and Garcia-Verdugo, 2002; Alvarez-Buylla and Lim, 2004; Erickson et al., 1998). It is the latter that is directly relevant to this research. There are several progenitor cell stages. 1) Type –1 cells have a radial-glia-like morphology and express the astrocytic antigen glia fibrillary acidic protein (GFAP). Type-1 cells can also give rise to GFAP positive S-100ß positive astrocytes. The daughters of Type-1 cells either express S-100ß or neuron-specific doublecortin (DCX, expressed in immature neurons), but not both. 2) Type-2 cells give the appearance of migrating along the SGZ (Kempermann, et al., 2004). They are GFAP negative. 3) Type-3 cells represent a further maturation developing a much more rounded nucleus and cell body. They are still proliferative and express DCX. Type-3 cells after further development become immature granule cells that are postmitotic. Mature granule cells are present 2-3 weeks after becoming postmitotic. They stop expressing DCX and calretinin, instead they express calbindin, and the postmitotic neuronal marker NeuN.

The number of new cells that survive to maturity is a small fraction of the total that are born. The majority of expansion of this population occurs due to proliferation of cells after Type-1. (Brandt, et al., 2003). It is this huge surplus of proliferating cells neuronal lineage precursors that offers the opportunity to restore lost granule cells in this research.

Regulation of the rate of neurogenesis is controlled by a variety of behaviorally derived factors and endogenous regulators. Wheel-running increases the rate of proliferation of Type-2 cells, but not Type-3 cells (Kempermann, et al., 2004a; Kronenberg, et al., 2003). Environmental enrichment can dramatically increase the number of new neurons in DG, but it may not increase proliferation of any of the cell types.

Environmental enrichment may primarily increase the proportion of neuronal precursors that are selected to survive to granule cell maturity (Kronenberg, et al., 2003).

A variety of neurotransmitters, hormones, modulators, and neurotrophins affect adult HPC neurogenesis. antagonists at the NMDA receptor increase proliferation (Seri and Alvarez-Bullya, 2002). Not all agents that affect proliferation have a similar effect on long-term survival of new cells. For example, estrogen, which decreases extracellular glutamate concentrations in DG via increase NMDA receptor number, increases proliferation but does not have a reliable effect on long-term survival (Seri and Alvarez-Bullya, 2002). Certain serotonergic agonists increase proliferation and long-term survival of new granule cells. (Banasr, et al., 2004; Djavadian, 2004; Malberg et al., 2000). The basis for the stimulation of proliferation and survival in the dentate is unclear. CORT levels also determine proliferation rate. Elevating circulating CORT level decreases both proliferation and long-term survival; ADX increases proliferation (Cameron and Gould, 1994, 1996). Several neurotrophins and related factors have a potent effect in accelerating proliferation. These include epidermal growth factor (EGF), brain-derived neurotrophic factor (BDNF), insulin-like growth factor (IGF-2), fibroblast growth factor (FGF-2 or bFGF), vascular endothelial growth factor (VEGF), erythropoietin (EPO), and sonic hedgehog (shh) (Alvarez-Buylla and Lim, 2004). It should be noted that very little or no work on any combinations of these proliferation/neuronal survival factors with or without behavioural neurogenic stimuli has been carried out in vivo with HPC neurogenesis.

Are new granule cells functional? It is clear that adult neurogenesis produces cells that are in the correct locations, express granule cell specific antigens, normal dendritic arbour, and extend mossy fiber-type axons into CA3, all characteristics of mature granule cells (Kempermann, et al., 2004b; Stanfield and Trice, 1988). It was demonstrated in HPC slices that weeks after birth, new granule cells acquired the electrophysiological properties of normal older granule cells (DG. Van Praag et al. 2002).

In summary, there are several practical routes to enhance proliferation and long-term survival of new neurons in the HPC DG.

II-4. Significance and Implications.

The goal of this project is to systematically evaluate whether neurogenesis can repair damaged brain circuitry. Indeed, it is the case. The results of this project is pivotal for research aimed at reversing the effects of neurological degenerative diseases and brain injury. Also, this project, by evaluating the effects of damage restricted to the DG on memory, contributes to a greater understanding of general hippocampal function.

III. Materials and Methods

Animals: Male Long Evan Rats , 3 months of age, are used for surgery. Animals are housed in pairs before surgery. They are under normal 12 hour light cycle with food and water ad libitum. All animal procedures will

be performed according to protocols approved by appropriate Animal Care and Use Committees.

Surgery: bilateral adrenal removal (ADX) will be performed at age of 3 month. Sham controls will undergo the same surgery as ADX rats, except no adrenal tissue will be removed. At all times during all experiments rats will be housed in pairs or in condoes (6 animals) with continual access to water and 0.9% saline. All rats will be weighed each week. At the end of the experiment and before initiating CORT replacement, a blood sample will be taken and plasma CORT will be measured using a radioimmunoassay procedure (Maclennan et al., 2003). Unsuccessful ADX rats will not participate in further experimentation (McCormick et al., 1997). Promote Neurogenesis and survival: Factors that can promote neurogenesis were carried in an osmotic pump which is surgically set up under the skin and the treatment are infused into ventricle. Environmental enrichment was used to increase the rate of new neuron survival.

Measuring Neurogenesis: Animals were perfused with 4% paraformaldehyde after done with behavior tests. Section of the hippocampus are stained with neuron maker (NeuN) to determine the total number of granule cells. Antigen-positive cells were counted in Z-sectioned sections acquired using a Zeiss Confocal microscope, only in the granular layers and adjacent subgranular zones. Proliferation after ADX, ADX-CORT replacement, and all of our treatments were quantified immunohistochemically in two ways: with BrdU and with Ki67. Ki67 is an antigen expressed by cycling cells for approximately 12-24 hr. Numbers of new neural precursors were measured by labeling the DCX antigen and the number of these that are actively cycling were assessed using DCX + Ki67 double-labeling. the number of new mature neurons were assessed in separate groups of rats who receive BrdU injections. BrdU+NeuN and BrdU+S-100ß double-labeling were applied to quantify new mature neurons and glia.

Behavioral Tests: (1) Morris water task: rats receive 8 trials per day with a hidden platform. Rats were released from the 4 cardinal compass points twice each day in a pseudorandom sequence. On odd-numbered days the platform is repositioned in a new hidden location, remaining there for all trials of that day, and on even-numbered days the platform remains in the same location as the previous day. On every trial we automatically record time to find the platform, swim path length, heading error, proportion of path in each quadrant and annulus, and swim speed. (2) Fear conditioning to context task: If rats have had the opportunity to explore the novel context the day before for a few minutes, then the immediate shock creates a strong conditioned fear of the context. Thus, the rat must have, at the time of entry into the shock situation, retrieved the memory of the explored context and thereby formed a conditioned response to it (Rudy et al., 2002; Rudy and O’Reilly, 1999). This procedure has been shown to be sensitive to HPC damage. (3) Object recognition test: Two different shapes of boxes were used. Each one contains two identical objects inside. Animals spend some time to get familiar with the box and associate the box with certain object. Then one of the object was replaced with a different type of object. A normal animal will spend more time on the new object. The time animals spend on two different objects were compared between treated animals and control animals.

Figure 2. ADX animals have behavior deficit in compare to sham animals no matter if they were kept in a cage or in the enriched environment. After the cocktail treatment in conjunction with environmental enrichment, the performance of ADX animals in the behavior test similar to sham.

Figure 3. The volumes of dentate gyrus were reduced in ADX animals due to neuron death. The lost granule cells were repopulated after the cocktail treatment. The volumes of dentate gyrus were similar to sham animals after the cocktail treatment.

From my studies (figure1and 2 ), I have shown that ADX caused granule cell death in the hippocampus and lead to behavior deficit. The behavior deficit was reversed after re-population of the granule cells by the cocktail treatment (Lai et al 2011a, submitted). The volume of dentate gyrus in the ADX animals were repopulated as shown in figure 3

Recently, we have established the primary cell culture system for the neural progenitor cells in the adult hippocampus. Figure 4 shows the purified progenitor cells 2 days and 10 days after cultured in the dish

respectively. We used this primary cell culture system to do the primary screen for the candidate which promot neurogenesis . And study the transition of neural progenitor cells and cell-cell interaction in vitro.

In general, I have characterized the behavioral and physiological deficits after targeted degeneration of hippocampus granule cells. The ADX animals show behavior deficit in morris water maze, contextual fear conditioning, and object-context assocition (Lai et. al., 2011b submitted). After the cocktail treatment, we were able to reverse the behavior deficit possibly through enhancement of neurogenesis and integration of new neurons (Lai et. al., 2011b submitted). At current stage, we would like to screen for more potential treatment and try to understand the mechanism behind the treatment. If the results are promissing, clinical application for neurodegenerative disorder is the ultimate goal.

V. Referances

Alvarez-Buylla A. and Garcia-Verdugo JM. (2002)Neurogenesis in adult subventricular zone. J Neurosci. 22(3):629-34

Alvarez-Buylla, A and Daniel A. Lim (2004). For the Long Run: Maintaining Germinal Niches in the Adult Brain. Neuron, 41, 683–686.

Banasr M, Hery M, Printemps R, Daszuta A (2004). Serotonin- induced increases in adult cell proliferation and neurogenesis are mediated through different and common 5-HT receptor subtypes in the dentate gyrus and the subventricular zone. Neuropsychopharmacol 29, 450-460.

Brandt MD, Jessberger S, Steiner B, Kronenberg G, Reuter K, Bick-Sander A, Von der Behrens W, Kempermann G. (2003). Transient calretinin expression defines early postmitotic step of neuronal differentiation in adult hippocampal neurogenesis of mice. Mol Cell Neurosci 24, no.3, 603-613.

Cameron, HA; Gould, E (1994). Adult neurogenesis is regulated by adrenal steroids in the dentate gyrus. Neurosci 61, 203-209.

Cameron, H; Gould, E (1996). Distinct populations of cells in the adult dentate gyrus undergo mitosis or apoptosis in response to adrenalectomy. J Comp Neurol 369, 56-63

Czurko, A; Czeh, B; Seress, L; Nadel, L; Bures, J (1997). Severe spatial navigation deficit in the Morris water maze after single high dose of neonatal x-ray irradiation in the rat. Proc Nat Acad Sci USA 94, 2766-2771.

Djavadian, RL (2004). Serotonin and neurogenesis in the hippocampal dentate gyrus of adult mammals. Acta Neurobiol Exp 2004, 64: 189-200.

Dudchenko, PA; Wood, ER; Eichenbaum, H (2000). Neurotoxic hippocampal lesions have no effect on odor span and little effect on odor recognition memory but produce significant impairments on spatial span, recognition, and alternation. J Neurosci 20, 2964-2977.

Eriksson PS, Perfilieva E, Bjork-Eriksson T, Alborn A-M, Nordborg C, Peterson DA, Gage FH (1998) Neurogenesis in the adult human hippocampus. Nat Med 4: 1313-1317.

Gilbert, PE; Kesner, RP; Lee, I (2001). Dissociating hippocampal subregions: A double dissociation between dentate gyrus and CA1. Hippocampus, 11, 626-636.

Helene, J; Jason, Y; Elisabeth, A; Patricia, MP; Sarah, S; Luc, G; Sophie, C; Eliane, M; Jean-Christophe, C (2003). Transplantation of neurospheres after granule cell lesions in rats: cognitive improvements despite no long-term immunodetection of grafted cells. Behav Brain Res 143, 177-191.

Jeltsch, H; Bertrand, F; Lazarus, C; Cassel, JC (2001). Cognitive performances and locomotor activity following dentate granule cell damage in rats: Role of lesion extent and type of memory tested. Neurobiol Learn Mem 76, 81-105.

Kempermann, G Jessberger, S Steiner, B Kronenberg, G (2004a). Milestones of neuronal development in the adult hippocampus. Trends in Neurosci 27, 447-452.

Kempermann, G Wiskott, L Gage, FH (2004b) Functional significance of adult neurogenesis. Curr Opin Neurobiol 14, 186-191.

Kim JJ, Fanselow M. (1992). Modality-specific retrograde amnesia of fear. Science 256:675-676.

Kronenberg, G. et al. (2003) Subpopulations of proliferating cells of the adult hippocampus respond differently to physiologic neurogenic stimuli. J. Comp. Neurol. 467, 455–463

Lai, G.J., S. Spanswick, R. Sutherland, Behavior deficit, neurogenesis, and functional recovery after granule cell death in the hippocampus. 2011a, submitted

Lai, G.J., S. Spanswick, R. Sutherland, Behavioral deficits induced by granule cell death in the hippocampus after adrenalectomy. 2011b, submitted

Lee, I Kesner, RP (2004a). Differential contributions of dorsal hippocampal subregions to memory acquisition and retrieval in contextual fear-conditioning. Hippocampus 14, 301-310.

Lee, I Kesner, RP (2004b). Encoding versus retrieval of spatial memory: Double dissociation between the dentate gyrus and the perforant path inputs into CA3 in the dorsal hippocampus. Hippocampus, 14, 66-76. Maclennan, KM; Zheng, YW; Sheard, PW; Williams, SM; Darlington, CL; Smith, PF (2003). Adrenalectomy-induced cell death in the dentate gyrus: Further characterisation using TUNEL and effects of the Ginkgo biloba extract, EGb 761, and ginkgolide B. Hippocampus, 13, 212-225.

Malberg JE, Eisch AJ, Nestler EJ, Duman RS (2000). Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus. J Neurosci 20: 9104–9110.

McCormick, CM; McNamara, M; Mukhopadhyay, S; Kelsey, JE. (1997). Acute corticosterone replacement reinstates performance on spatial and nonspatial memory tasks 3 months after adrenalectomy despite degeneration in the dentate gyrus. Behav Neurosci 111, 518-531.

Morris, R.G.M., Garrud, P., Rawlins, J., O’Keefe, J. (1982) Place navigation is impaired in rats with hippocampal lesions. Nature 297:681-683.

Phillips RG, LeDoux JE. Lesions of the dorsal hippocampal formation interfere with background but not foreground contextual fear conditioning. Learn Mem 1994;1:34-45.

Prusky, GT; Douglas, RM; Nelson, L; Shabanpoor, A; Sutherland, RJ (2004a). Visual memory task for rats reveals an essential role for hippocampus and perirhinal cortex. Proc Nat Acad Sci USA 101, 5064-5068.

Rudy JW, Barrientos RM, O'Reilly RC. The hippocampal formation supports conditioning to memory of a context. Behav Neurosci 2002;116:530-538.

Rudy JW, O’Reilly RC. (1999). Contextual fear conditioning, conjunctive representations, pattern completion, and the hippocampus. Behav Neurosci 113: 67-880.

Seri, B Alvarez-Buylla, A (2002). Neural stem cells and the regulation of neurogenesis in the adult hippocampus. Clin Neurosci Res 2, 11-16.

Shetty, AK; Turner, DA (1996). Development of fetal hippocampal grafts in intact and lesioned hippocampus Prog in Neurobiol. 50, 597-653.

Sloviter, RS Valiquette, G Abrams, GM Ronk, EC Sollas, AL Paul, LA Neubort, S (1989). Selective loss of hippocampal granule cells in the mature rat brain after adrenalectomy. Science, 243, 535-538.

hippocampal granule cell degeneration in the rat: Apoptosis in the adult central nervous system. J Comp Neurol 330, 337-351.

Sloviter, RS Sollas, AL Dean, E Neubort, S (1993b) Adrenalectomy-induced granule cell degeneration in the rat hippocampal dentate gyrus: Characterization of an in vivo model of controlled neuronal death. J Comp Neurol 330, 324-336.

Sousa, N Madeira, MD Paula-Barbosa, MM (1997). Structural alterations of the hippocampal formation of adrenalectomized rats: An unbiased stereological study. J Neurocytol 26, 423-438.

Stanfield BB, Trice JE (1988). Evidence that granule cells generated in the dentate gyrus of adult rats extend axonal projections. Exp Brain Res 72, 399-406.

Sutherland, R. J. & McDonald, R. J. (1990). Hippocampus, amygdala, and memory deficits in rats. Behavioural Brain Research, 37, 57-79.

Sutherland, R. J., Whishaw, I. Q., & Kolb, B. (1983). A behavioural analysis of spatial localization following electrolytic, kainate-, or colchicine-induced damage to the hippocampal formation in the rat. Behavioural Brain Research, 7, 133-153.

Tandon, P Barone, S Drust, EG Tilson, HA (1991). Long-term behavioral and neurochemical effects of intradentate administration of colchicine in rats. Neurotoxicol 12, 67-78.

Turner, DA; Shetty, AK (2003). Clinical prospects for neural grafting therapy for hippocampal lesions and epilepsy. Neurosurgery 52, 3, 632-641.

Van Praag H, Schinder AF, Christie BR, Toni N, Palmer TD, Gage FH (2002). Functional neurogenesis in the adult hippocampus. Nature 415:1030-1034.

Xavier, GF; Oliveira, FJB; Santos, AMG (1999). Dentate gyrus-selective colchicine lesion and disruption of performance in spatial tasks: Difficulties in "place strategy" because of a lack of flexibility in the use of environmental cues? Hippocampus 9, 668-681.

國科會補助專題研究計畫成果報告自評表

請就研究內容與原計畫相符程度、達成預期目標情況、研究成果之學術或應用價

值（簡要敘述成果所代表之意義、價值、影響或進一步發展之可能性）、是否適

合在學術期刊發表或申請專利、主要發現或其他有關價值等，作一綜合評估。

1.

請就研究內容與原計畫相符程度、達成預期目標情況作一綜合評估

þ

達成目標

□ 未達成目標（請說明，以 100 字為限）

□ 實驗失敗

□ 因故實驗中斷

□ 其他原因

說明：

2.

研究成果在學術期刊發表或申請專利等情形：

論文：□已發表 þ未發表之文稿 □撰寫中 □無

專利：□已獲得 □申請中 □無

技轉：□已技轉 □洽談中 □無

其他：（以 100 字為限）

附件二

3.

請依學術成就、技術創新、社會影響等方面，評估研究成果之學術或應用價

值（簡要敘述成果所代表之意義、價值、影響或進一步發展之可能性）（以

500

字為限）

海馬迴是腦中負責學習與記憶的重要部位，也是許多神經疾病的發病點，像是阿茲海莫 症，失憶症，中風，癲癇，長期壓力等等。如果能找到方法來修復腦部的病變，將可造福很 多人。在成人或成鼠的大腦裡有兩個地方可以持續的婧進行神經細胞新生，海馬迴的粒細胞 是其中一個。 移除腎上腺可以很專一的造成海馬迴粒細胞死亡而不影響腦部任何其他區 域，我以此作為研究系統來建立神經細胞新生並恢復腦部功能的系統，並借此更加了解神經 細胞新生並恢復腦部功能的機制。利用個體本身內生性”幹”細胞來新生細胞將可免除手術不 便，幹細胞來原的問題，以及異體移植所產生的排斥。本方法未來具臨床應用可行性。

國科會補助專題研究計畫項下出席國際學術會議心得報告

日期：100 年 10 月 1 日

一、參加會議經過:

Taos is a mountain city, a popular skiing area. It took me long travel to get there. The symposium started early each morning. There are many invited speakers every morning. I learned lots of outstanding research in this section. In the afternoon, there are couple hours break for social activity. In the evening, everyone back for dinner and poster section. The poster section finished at 10pm officially, but sometimes we stayed till midnight for interesting discussion.

二、與會心得：

I am glad that I found this meeting, adult neurogenesis, hosted by “keystone symposium”. “Adult neurogenesis” is the main theme of my current research. There are about 300 people attend the meeting. It’s big enough to cover all the respect of adult neurogenesis and yet small enough to get to know some researchers and line up for some international collaboration. I met 3 principle investigators that offer future collaboration (one for sending transgenic mice, one for transgenic rat planning, and one for the viral vector approach). Besides that, I talked to several researchers and lots of critical experimental tricks, which are not written in the textbook. And I learned new experimental design as well. Overall, it is a very good

計畫編號

NSC 98-2314-B-004-001-MY2

計畫名稱

壓力荷爾蒙與神經細胞之再生及死亡

出國人員

姓名

賴桂珍

服務機構

及職稱

國立政治大學神經科學所

會議時間

2011 年 1 月 9 日

至 2011 年 1 月 14

日

會議地點

Sagebrush Inn and Conference

Center, Taos, New Mexico, USA

會議名稱

(中文)成熟個體神經新生

(英文)Keystone Symposium: Ault Neurogenesis

發表論文

題目

(中文)海馬迴粒細胞死亡後索引發之行為缺陷及治療後之功能恢復

(英文)

Behavior deficit and functional recovery after granule cell death in the hippocampus

meeting and I will definitely attend the meeting again if there is another one coming in the next couple of years.

三、考察參觀活動(無是項活動者略)

四、建議

五、攜回資料名稱及內容

六、其他

Acceptance  of  abstract  submission    

寄件人 [email protected] 收 [email protected] 日期 2010 年 12 月 1 9:01

主旨 Keystone Symposia Poster Abstract information keystonesymposia.org

隱藏詳細資料 10/12/1

Wednesday, December 1, 2010 Guey-Jen Lai

[email protected]

Adult Neurogenesis (A5)

Dates: January 9 - January 14, 2011

Location: Sagebrush Inn and Conference Center, Taos, New Mexico Early Registration Deadline: November 10, 2010

Congratulations, your abstract has been accepted to this program. ---

Poster Number: 228

Poster Title: Behavior deficit and functional recovery after granule cell death in the hippocampus Authors: G Lai; S Spanswick; R Sutherland

Poster Size: 1 meter by 1 meter (3.3 feet by 3.3 feet) Session: Poster Session 2

Tuesday, January 11, 2011 ---

This acceptance allows your work to be shown to organizers, speakers and attendees of this meeting. Based on comments from our surveys, poster sessions are one of the most important aspects of the conference. Several collaborations have resulted from the informal discussions held at these sessions. Your presentation at the poster session is vital to the success of the meeting.

We also strongly recommend that you have your abstract viewable online to other registered delegates prior to the meeting. Reviewing the abstracts in advance allows attendees to maximize their time with each presenter. Instructions for making your abstract viewable are listed below under ONLINE VIEWING.

Important information about your presentation is listed below:

REGISTRATION

- Abstract Submission / Poster Acceptance does not guarantee registration.

- If you have not already registered for this meeting, please do so as soon as possible. - You may register at http://www.keystonesymposia.org/accounts/ListAccount.cfm

- Login to My Account and click the Register Yourself button.

- Oral presentations and short talks are to be determined by the organizers. Those selected will receive invitations from the Keystone Symposia office under separate cover.

- If selected for a Short Talk, you will still be expected to present your poster during the above assigned poster session.

POSTERS

- Each poster will have 1 meter by 1 meter (3.3 feet by 3.3 feet) of space available for display. - Your poster will be viewed from a distance of several feet; use bold type and large figures.

- Poster boards will be numbered.

- Thumbtacks and pushpins will be available for mounting your materials. - Poster sessions will be held from 7:30 PM - 10:00 PM.

- The poster must be removed by the end of the poster session. - Staff is not responsible for the safe return of your poster. - If your poster is oversized, your space cannot be guaranteed. ONLINE VIEWING

- You may view your abstract and upload revisions on our website until November 10, 2010.

- Our records show you have chosen to NOT have your abstract viewable to registered attendees. Other abstracts for this meeting will be viewable beginning December 9, 2010

- You may change whether your abstract is or is not viewable on our website.

- Use the My Account link at the top right of our website to login. Then click Edit Your Abstract to do the above.

- If you have any questions, please contact our office at +1 800-253-0685 or +1 970-262-1230, or

[email protected]. PUBLISHING

- Your abstract will be published in a book which will include all poster and speaker abstracts received for this meeting.

- This book will be distributed to all registered conferees upon check-in. - Abstract presenters will be referenced in the book.

We look forward to your participation in what promises to be an exciting meeting. Sincerely,

Keystone Symposia on behalf of

Jenny Hsieh, Fred H. Gage, Alejandro Fabian Schinder and Pierre-Marie Lledo, Organizers Adult Neurogenesis                                          

   

Abstract

Behavior deficit and functional recovery after granule cell death in the hippocampus

G.-J. LAI1,2, S. SPANSWICK , R. J. SUTHERLAND 1 1 Dept Neurons, Univ. Lethbridge, Lethbridge, AB, T1K 3M4,

1

Canada

2 _{Inst. Neurosci, National Chengchi Univ., Taipei, 116,}

Taiwan, R.O.C.,

The dentate gyrus subgranular zone of the hippocampus is the source of newly born granule cells. Bilateral removal of adrenal glands (adrenalectomy, ADX) leads to specific

granule cell death in the hippocampus without significant effects in other regions of the brain. Thus, we used this model to study regeneration of lost granule cells and its role in functional restoration of brain circuitry.

Many substances are known to accelerate neurogenesis, but there are few data regarding the restoration of functional brain network after increased neurogenesis. We demonstrate that treatment with sonic hedgehog and corticosterone supplement in combination with environmental enrichment can promote neurogenesis and restore the function of the lost granule cells. In object-context recognition tests, the behavior deficit caused by ADX was rescued by the treatment. The number of granule cells in treated ADX animals increased significantly in comparison with vehicle treated ADX rats.

This is the first model system for studying the

regeneration of selectively and progressively ablated neurons and recovery of cognitive function. This work provides an opportunity to evaluate endogenous neurogenesis promotion in adults for neuron degenerative disease treatment and also provides an opportunity to understand the function of hippocampal granule cells.

Supported by: CIHR Grant 45188, AHFMR Postdoctoral

Fellowship, CIHR/HSFC Focus on Stroke Research Award, NSC 98-2314-B-004-001-MY2

參加國際會議心得

I am glad that I found this meeting, adult neurogenesis, hosted by “keystone symposium”. “Adult neurogenesis” is the main theme of my current research. There are about 300 people attend the meeting. It’s big enough to cover all the respect of adult neurogenesis and yet small enough to get to know some researchers and line up for some international

collaboration. I met 3 principle investigators that offer future collaboration (one for sending transgenic mice, one for transgenic rat planning, and one for the viral vector approach). Besides that, I talked to several researchers and lots of critical experimental tricks, which are not written in the textbook. And I learned new experimental design as well. Overall, it is a very good meeting and I will definitely attend the meeting again if there is another one coming in the next couple of years.

國科會補助計畫衍生研發成果推廣資料表

日期:2012/01/27

國科會補助計畫

計畫名稱: 壓力荷爾蒙與神經細胞之再生及死亡 計畫主持人: 賴桂珍 計畫編號: 98-2314-B-004-001-MY2 學門領域: 幹細胞/再生生物醫學

無研發成果推廣資料

98 年度專題研究計畫研究成果彙整表

計畫主持人：賴桂珍 計畫編號：98-2314-B-004-001-MY2 計畫名稱：壓力荷爾蒙與神經細胞之再生及死亡 量化 成果項目 實際已達成 數（被接受 或已發表） 預期總達成 數(含實際已 達成數) 本計畫實 際貢獻百 分比 單位 備 註 （ 質 化 說 明：如 數 個 計 畫 共 同 成 果、成 果 列 為 該 期 刊 之 封 面 故 事 ... 等） 期刊論文 0 0 100% 研究報告/技術報告 0 0 100% 研討會論文 0 0 100% 篇 論文著作 專書 0 0 100% 申請中件數 0 0 100% 專利 已獲得件數 0 0 100% 件 件數 0 0 100% 件 技術移轉 權利金 0 0 100% 千元 碩士生 4 4 100% 博士生 0 0 100% 博士後研究員 0 0 100% 國內 參與計畫人力 （本國籍） 專任助理 0 0 100% 人次 期刊論文 0 3 100% 研究報告/技術報告 0 0 100% 研討會論文 2 2 100% 篇 論文著作 專書 0 0 100% 章/本 申請中件數 0 0 100% 專利 已獲得件數 0 0 100% 件 件數 0 0 100% 件 技術移轉 權利金 0 0 100% 千元 碩士生 0 0 100% 博士生 0 0 100% 博士後研究員 0 0 100% 國外 參與計畫人力 （外國籍） 專任助理 0 0 100% 人次

其他成果

(

無法以量化表達之成 果如辦理學術活動、獲 得獎項、重要國際合 作、研究成果國際影響 力及其他協助產業技 術發展之具體效益事 項等，請以文字敘述填 列。) 無 成果項目 量化 名稱或內容性質簡述 測驗工具(含質性與量性) 0 課程/模組 0 電腦及網路系統或工具 0 教材 0 舉辦之活動/競賽 0 研討會/工作坊 0 電子報、網站 0 科 教 處 計 畫 加 填 項 目 計畫成果推廣之參與（閱聽）人數 0

國科會補助專題研究計畫成果報告自評表

請就研究內容與原計畫相符程度、達成預期目標情況、研究成果之學術或應用價

值（簡要敘述成果所代表之意義、價值、影響或進一步發展之可能性）

、是否適

合在學術期刊發表或申請專利、主要發現或其他有關價值等，作一綜合評估。

1. 請就研究內容與原計畫相符程度、達成預期目標情況作一綜合評估

■達成目標

□未達成目標（請說明，以 100 字為限）

□實驗失敗

□因故實驗中斷

□其他原因

說明：

2. 研究成果在學術期刊發表或申請專利等情形：

論文：□已發表 ■未發表之文稿 □撰寫中 □無

專利：□已獲得 □申請中 ■無

技轉：□已技轉 □洽談中 ■無

其他：（以 100 字為限）

有三篇文稿準備發表

3. 請依學術成就、技術創新、社會影響等方面，評估研究成果之學術或應用價

值（簡要敘述成果所代表之意義、價值、影響或進一步發展之可能性）（以

500 字為限）

本研究利用將腎上腺移除引發海馬迴中粒細胞死亡的模式來探討壓力荷爾蒙對海馬迴粒 細胞存活的重要性，並尋找促進成鼠神經細胞新生，建立適當連結，以恢復受損海馬迴功 能。 移除腎上腺可以很專一的造成海馬迴粒細胞死亡而不影響腦部任何其他區域，我以此作為 研究系統來進行神經細胞新生並恢復腦部功能，並借此更加了解神經細胞新生並恢復腦部 功能的機制。利用個體本身內生性’幹細胞’來再生細胞將可免除手術不便，幹細胞來源 的問題，以及異體移植所產生的排斥。本方法未來具臨床應用可行性。