蛋白偶联受体GPR40在缺血性成年猴海马的表达

目的 利用猴短暂性全脑缺血模型,研究蛋白偶联受体40(GPR40)在海马的表达,从而探讨GPR40受体在缺血性卒中的作用.方法 参照Yamashima建立成年猴短暂性全脑缺血模型,24只猴随机数字表法分成4组,即对照组、缺血后4 d组(d4)、缺血后9 d组(d9)和缺血后15 d(d15),采用Western印迹和免疫荧光在蛋白质水平观察GPR40的表达.结果 Western blotting结果显示CA1区域GPR40在短暂性全脑缺血后表达逐渐下降(15 d内);而在DG区域表达升高,与对照组比较有统计学意义(P<0.05).免疫荧光分析显示CA1区域与GPR40共表达的神经元细胞数逐渐下降.到d15约下降50%;而DG区域则无明显变化,邻近SGZ区域与GPR40共表达的星形细胞数量迅速升高,d4升高约2.5倍.结论 脑缺血后海马CA1和DG两区域GPR40表达差异.特别SGZ星形细胞GPR40表达升高,提示可通过增加GPR40配体中长链不饱和脂肪酸如DHA来改善脑缺血后海马神经元的损伤.     

Expression of matrix metalloproteinases in the neurogenic niche of the adult monkey hippocampus after ischemia

Matrix metalloproteinases (MMPs), zinc-dependent endopeptidases capable of remodeling extracellular matrix and regulating cellular signals, have been implicated in various neurological functions and diseases. However, the role of MMPs in the adult neurogenesis still remains to be clarified, particularly in the primate. Here, we studied differential expression of MMP9/2 in the neurogenic niche of the hippocampal dentate gyrus (DG) after transient global brain ischemia in young adult macaque monkeys. Zymography demonstrated biphasic upregulation of MMP9 in acute (Days 1-3) and delayed (Days 7-15) phases of postischemic reaction, whereas the level of MMP2 was elevated only in the delayed phase. Immunofluorescence histochemistry showed that MMP9 and MMP2 colabeled with markers of endothelial cells, astrocytes, and newborn neurons in the subgranular zone (SGZ) of the DG, and also that the percentage of coexpression significantly increased in the delayed postischemic phase, as compared with controls. However, colabeling with different cell selective markers reached its peak at different time points, i.e., with endothelial cells on Day 7, whereas with astrocytes and newborn neurons on Day 15, respectively. MMPs were localized both in the perikarya and dendrites in the newborn neurons. In conclusion, MMP9/2 expression was regulated in a cell- and time-specific manner in hippocampal neurogenic niche of adult primates.     

Cellular localization of epidermal‐type and brain‐type fatty acid‐binding proteins in adult hippocampus and their response to cerebral ischemia

This study aimed at an analysis of expression of epidermal‐type and brain‐type fatty acid‐binding proteins (E‐FABP and B‐FABP, also called FABP5 and FABP7, respectively) in adult hippocampus and their potential value as neuroprotective factors after ischemic brain damage in monkey model. The immunostaining and Western blotting results show that FABP5 was mainly expressed in neurons, whereas FABP7 was primarily expressed in astrocytes and progenitors of the subgranular zone (SGZ). Interestingly, FABP5 expression in neurons increased in cornu Ammonis 1 (CA1) and remains stable within dentate gyrus (DG) after ischemia; FABP7 expression increased within both CA1 and SGZ. This indicates a potential role for FABP5 and FABP7 in intracellular fatty acid transport within different neural cells. The change in FABP5–7 expression within CA1 and DG of the adult postischemic hippocampus was compatible with previous findings of downregulation in CA1 neurons and upregulation in SGZ progenitor cells after ischemia. Altogether, the present data suggest that polyunsaturated fatty acids, such as docosahexaenoic acid, may act via FABP5 or 7 to regulate adult postischemic hippocampal neuronal antiapoptosis or neurogenesis in primates. © 2009 Wiley‐Liss, Inc.     

DHA介导GPR40转染PC12细胞内的钙离子动员

目的 构建GPR40基因质粒转染PC12细胞,用DHA干预后测定细胞内Ca2+浓度的变化,从而探讨DHA通过GPR40信号途径介导Ca2+上调的作用机制.方法 将PC12细胞分成4组,分别为未转染组.空载体转染组,GPR40基因-pCruz载体转染组及GPR40基因-pCruz载体转染+阻制剂Xestospongin C组;构建大鼠GPR40质粒并转染PC12细胞.采用RT-PCR和Western blot方法 从mRNA和蛋白质水平观察GPR40的表达.DHA(10 μmol/L)干预后,测定细胞内Ca2+浓度.结果 DHA干预后,未转染组和空转染组PC12细胞内Ca2+浓度没有受到明显影响;GPR40基因-pCruz载体转染组细胞内的Ca2+浓度明显增加,且这种作用不受细胞外Ca2+浓度的影响:添加阻滞剂组Ca2+浓度变化也不明显.结论 DHA能动员GPR40基因转染的PC12细胞内Ca2+浓度且这种作用完全能被IP3受体特异性阻止剂Xestospongin C所阻断,提示DHA可能通过GPR40信号途径动员细胞内Ca2+浓度并因此改善神经功能.     

Origin of the dopaminergic innervation of adult neurogenic areas

Adult neurogenesis, a specific form of brain plasticity in mammals occurring in the subventricular zone (SVZ) and the subgranular zone (SGZ), is subject to complex regulations. Dopamine has been identified as a regulatory factor in rodents, nonhuman primates, and humans. We therefore studied in detail the origin of the dopaminergic innervation to the SVZ and SGZ in rodents. Postmortem anterograde and retrograde tracing combined with immunofluorescence procedures indicate a topographic organization of midbrain dopaminergic projections toward the SVZ. The substantia nigra pars compacta (SNc) projects specifically into the rostral migratory stream, the anterior SVZ, and the dorsal part of the medial and posterior SVZ. In contrast, the ventral tegmental area (VTA) innervates specifically the ventral part of the medial and posterior SVZ. Both VTA and SNc dopaminergic neurons innervate homogenously the lower and upper blade of the dentate gyrus, including the SGZ. Based on these findings, we suggest that the neurogenic areas are in a position to be differentially affected by region‐specific changes in dopamine signaling, the functional implication of which must be further elucidated. J. Comp. Neurol. 522:2336–2348, 2014. © 2014 Wiley Periodicals, Inc.     

Newly generated granule cells show rapid neuroplastic changes in the adult rat dentate gyrus during the first five days following pilocarpine-induced seizures

Long-term neuroplastic changes to dentate granule cells have been reported after seizures and were shown to contribute to recurrent excitatory circuitry. These changes include increased numbers of newborn granule cells, sprouted mossy fibers, granule cell layer dispersion, increased hilar ectopic granule cells and formation of hilar basal dendrites on granule cells. The goal of the current study was to determine the acute progression of neuroplastic changes involving newly generated granule cells after pilocarpine-induced seizures. Doublecortin (DCX) immunocytochemical preparations were used to examine the newly generated granule cells 1-5 days after seizures were induced. The results showed that there are rapid neuroplastic changes to the DCX-labeled cells. At 1 day after seizures were induced, there were significant increases in the percentage of DCX-labeled cells with hilar basal dendrites and in the progenitor cell population. At 2 days after seizures were induced, an increase in the thickness of the layer of DCX-labeled cells occurred. At 3 days after seizures were induced, the number of DCX-labeled cells was significantly increased. At 4 days after seizures were induced, developing synapses were observed on DCX-labeled hilar basal dendrites. Thus, newly generated granule cells in the adult dentate gyrus display neuroplastic changes by 1 day after pilocarpine-induced seizures and further changes occur to this population of cells in the subsequent 4 days. The presence of synapses, albeit developing ones, on hilar basal dendrites during this period indicates that newly generated granule cells become rapidly incorporated into dentate gyrus circuitry following seizures.     

Astrocyte fatty acid binding protein‐7 is a marker for neurogenic niches in the rat hippocampus

Recent research has determined that newborn neurons in the dentate gyrus of the hippocampus of the macaque are frequently adjacent to astrocytes immunoreactive for fatty acid binding protein‐7 (FABP7). To investigate if a similar relationship between FABP7‐positive (FABP7+) astrocytes and proliferating cells exists in the rodent brain, sections of brains from juvenile rats were stained by immunohistochemistry to demonstrate newborn cells (antibody to Ki67 protein) and FABP7+ astrocytes. In rat brains, FABP7+ astrocytes were particularly abundant in the dentate gyrus of the hippocampus and were frequently close to dividing cells immunoreactive for Ki67 protein. FABP7+ astrocytes were also present in the olfactory bulbs, arcuate nucleus of the hypothalamus, and in the dorsal medulla subjacent to the area postrema, sites where more modest numbers of newborn neurons can also be found. These data suggest that regional accumulations of FABP7+ astrocytes may represent reservoirs of cells having the potential for neurogenesis. Because FABP7+ astrocytes are particularly abundant in the hippocampus, and since the gene for FABP7 has been linked to Alzheimer's disease, age‐related changes in FABP7+ astrocytes (mitochondrial degeneration) may be relevant to age‐associated disorders of the hippocampus. © 2013 Wiley Periodicals, Inc.     

Maturational sequence of newly generated neurons in the dentate gyrus of the young adult rhesus monkey

The generation of new neurons in the hippocampal dentate gyrus of adult mammals has been characterized in rodents, but the details of this process have not been described in the primate. Eleven young adult rhesus monkeys were given an injection of the DNA synthesis phase marker bromodeoxyuridine (BrdU) and killed at varying survival intervals (2 hours to 98 days). The immature neuronal marker TUC-4 (TOAD/Ulip/CRMP-4) was used to define three stages of morphological maturation. Stage I neurons had small somata and lacked dendrites. Stage II neurons had larger somata and short dendrites. Stage III neurons were similar in size to mature granule cells and had branching dendrites that extended into the molecular layer. Examination of TUC-4-positive immature neurons colabeled with BrdU indicated that stage I neurons first appeared 2 days after BrdU injection, stage II neurons at 14 days, and stage III neurons at 35 days. Electron microscopy of TUC-4-labeled cells showed that stage I cells had ultrastructural features of immature neurons, whereas stage III neurons were similar to mature granule cells and formed synapses in the molecular layer. This suggests that stage III neurons could potentially integrate into the circuitry of the dentate gyrus. This study shows that the maturational sequence for new neurons in the adult monkey is similar to that of the adult rodent; however, maturation takes a minimum of 5 weeks in the monkey, which is substantially longer than what has been reported in rodents.     

An ultrastructural characterization of the newly generated cells in the adult monkey dentate gyrus

Recent studies of adult neurogenesis in the hippocampus have focused on the maturational sequence and on the identification of the neural stem cell in the adult brain. Ultrastructural verification of cell type and marker expression has become increasingly important in this research, yet no standards exist for the identification of adult generated cells in the hippocampus. In this study, six adult rhesus monkeys were used, four of which were given an injection of the DNA-synthesis phase marker bromodeoxyuridine (BrdU) and perfused 2 days, 3 weeks, or 6 weeks later. The ultrastructural features of BrdU labeled cells in the dentate gyrus were determined. The characteristics of the different types of BrdU labeled cells were then used to find similar, but unlabeled, immature cells in tissue routinely prepared for electron microscopy. This enabled optimal characterization of the ultrastructural features of the newly generated cells. The results demonstrate that immature neurons, immature astrocytes, and oligodendrocyte progenitor cells can be reliably distinguished by ultrastructural features, without immunohistochemical processing.     

Essential role of tau phosphorylation in adult hippocampal neurogenesis

An increased hippocampal neurogenesis has been observed in Alzheimer disease (AD), the most common neurodegenerative disorder characterized with accumulation of β‐amyloid (Aβ) and hyperphosphorylated tau (p‐tau). Studies in transgenic mouse models suggest that the amyloidosis suppresses adult neurogenesis. Although emerging evidence links tau to neurodevelopment, the direct data regarding tau phosphorylation in adult neurogenesis is missing. Here, we found that the immature neurons, identified by doublecortin (DCX) and neurogenic differentiation factor (neuroD), were only immunoreactive to p‐tau but not to the non‐p‐tau in adult rat brain and human patients with AD, and the p‐tau was coexpressed temporally and spatially with DCX and neuroD in the hippocampal dentate gyrus (DG) of the rat brains during postnatal development. A correlative increase of immature neuron markers and tau phosphorylation was induced in rat hippocampal DG by upregulating glycogen synthase kinase‐3 (GSK‐3), a crucial tau kinase, and the increased neurogenesis was due to an enhanced proliferation but not survival or differentiation of the newborn neurons. The hippocampal neurogenesis was severely impaired in tau knockout mice and activation of GSK‐3 in these mice did not rescue the deficits. These results reveal an essential role of tau phosphorylation in adult hippocampal neurogenesis. It suggests that spatial/temporal manipulation of tau phosphorylation may be compensatory for the neuron loss in neurological disorders, including AD. © 2009 Wiley‐Liss, Inc.     

The dynamics of adult neurogenesis in human hippocampus

The phenomenon of adult neurogenesis is now an accepted occurrence in mammals and also in humans.At least two discrete places house stem cells for generation of neurons in adult brain. These are olfactory system and the hippocampus. In animals, newly generated neurons have been directly or indirectly demonstrated to generate a significant amount of new neurons to have a functional role. However, the data in humans on the extent of this process is still scanty and such as difficult to comprehend its functional role in humans. This paper explores the available data on as extent of adult hippocampal neurogenesis in humans and makes comparison to animal data.     

Characterization of the Netrin/RGMa receptor neogenin in neurogenic regions of the mouse and human adult forebrain

In the adult rodent forebrain, astrocyte‐like neural stem cells reside within the subventricular zone (SVZ) and give rise to progenitors and neuroblasts, which then undergo chain migration along the rostral migratory stream (RMS) to the olfactory bulb, where they mature into fully functional interneurons. Neurogenesis also occurs in the adult human SVZ, where neural precursors similar to the rodent astrocyte‐like stem cell and neuroblast have been identified. A migratory pathway equivalent to the rodent RMS has also recently been described for the human forebrain. In the embryo, the guidance receptor neogenin and its ligands netrin‐1 and RGMa regulate important neurogenic processes, including differentiation and migration. We show in this study that neogenin is expressed on neural stem cells (B cells), progenitor cells (C cells), and neuroblasts (A cells) in the adult mouse SVZ and RMS. We also show that netrin‐1 and RGMa are ideally placed within the neurogenic niche to activate neogenin function. Moreover, we find that neogenin and RGMa are also present in the neurogenic regions of the human adult forebrain. We show that neogenin is localized to cells displaying stem cell (B cell)‐like characteristics within the adult human SVZ and RMS and that RGMa is expressed by the same or a closely apposed cell population. This study supports the hypothesis that, as in the embryo, neogenin regulates fundamental signalling pathways important for neurogenesis in the adult mouse and human forebrain. J. Comp. Neurol. 518:3237–3253, 2010. © 2010 Wiley‐Liss, Inc.     

Decline in adult neurogenesis during aging follows a topographic pattern in the mouse hippocampus

In the rodent brain, diverse functions are topographically distributed within the hippocampus. For instance, the dorsal (septal) hippocampus is involved in spatial memory, whereas the ventral (temporal) hippocampus is related to emotion and anxiety. Accumulating evidence shows that age-dependent decline in hippocampal neurogenesis is associated with impairments of these functions. However, little is known about whether the decline in dentate granule cell production during aging follows a topographic pattern. Here we quantitatively estimated specific populations of adult-born cells in young adult and middle-aged mice by using endogenous markers and determined whether age-dependent reductions in adult neurogenesis exhibited topographic differences. The numerical densities (NDs) of putative primary progenitors, intermediate neuronal progenitors, and neuronal lineages were higher in the dorsal dentate gyrus (DG) than in the ventral DG both in young adult and in middle-aged mice, but the ratios of the NDs in the dorsal DG to the NDs in the ventral DG noticeably increased with age. The age-related reductions in the numbers of these populations were larger in the ventral DG than in the dorsal DG. By contrast, the NDs of glial lineages were higher in the ventral DG than in the dorsal DG during life, and the numbers of glial lineages showed no significant age-related changes. Our findings suggest that neurogenesis, but not gliogenesis, wanes faster in the ventral hippocampus than in the dorsal hippocampus during aging. Such age-related topographic changes in hippocampal neurogenesis might be implicated in memory and affective impairments in older people.     

Neurogenic marker abnormalities in the hippocampus in dementia with Lewy bodies

Dementia with Lewy bodies (DLB) is associated with alpha synuclein pathology and slowly progressive dementia. Progenitor abnormalities have previously been reported in the subventricular zone (SVZ) adjacent to the lateral ventricle. To evaluate changes in neural stem cells and progenitors in the hippocampal neurogenic niche, immunohistochemistry (IHC) using the neural stem cell markers Musashi 1, nestin, proliferating cell nuclear antigen (PCNA), doublecortin, and glial fibrillary acidic protein (GFAP) were examined in age‐matched control and DLB groups. Staining was quantified in the hippocampal SVZ, subgranular layer (SGL) and ependymal cell layer (EPL). There was a significant loss in DLB of Musashi 1 (P < 0.01) in all areas, an increase in PCNA in hippocampal SVZ (P = 0.01) and SGL (P = 0.05), and an increase in doublecortin in the hippocampal SVZ (P = 0.04) and EPL (P = 0.02). This is the first report of the changes in neurogenic markers in the hippocampal SVZ and EPL in DLB and may offer the potential for understanding disease pathology and in the devising of treatment. ©2010 Wiley‐Liss, Inc.     

Impact of melatonin receptor‐signaling on Zeitgeber time‐dependent changes in cell proliferation and apoptosis in the adult murine hippocampus

The hippocampus is subjected to diurnal/circadian rhythms on both the morphological and molecular levels. Certain aspects of cell proliferation in the adult hippocampus are regulated by melatonin and accompanied by apoptosis to ensure proper tissue maintenance and function. The present study investigated Zeitgeber time (ZT)‐dependent changes in cell proliferation and apoptosis in the adult murine hippocampus and their regulation by melatonin receptor type1 and type2 (MT1/2)‐mediated signaling. Adult melatonin‐proficient C3H/HeN mice and melatonin‐proficient (C3H/HeN) mice with targeted deletion of MT1/2 were adapted to a 12‐h light, 12‐h dark photoperiod and were sacrificed at ZT00, ZT06, ZT12, and ZT18. Immunohistochemistry for Ki67 and activated caspase‐3 in combination with different markers for the diverse cell types residing in the hippocampus served to identify and quantify proliferating and apoptotic cells in the hippocampal subregions. ZT‐dependent changes in cell proliferation and apoptosis were found exclusively in the subgranular zone (SGZ) and granule cell layer (GCL) of melatonin‐proficient mice with functional MT1/2. Cell proliferation in the SGZ showed ZT‐dependent changes indicated by an increase of proliferating immature neurons during the dark phase of the 24‐h light‐dark cycle. Apoptosis showed ZT‐dependent changes in the SGZ and GCL indicated by an increase of apoptotic immature neurons at ZT06 (SGZ) and a decrease of immature and mature neurons at ZT18 (GCL). Our results indicate that ZT‐dependent changes in proliferation of immature neurons in the SGZ are counterbalanced by ZT‐dependent changes in apoptosis of immature and mature neurons in the SGZ and GCL exclusively in mice with functional MT1/2. Therefore, MT1/2‐mediated signaling appears to be crucial for generation and timing of ZT‐dependent changes in cell proliferation and apoptosis and for differentiation of proliferating cells into neurons in the SGZ. © 2017 Wiley Periodicals, Inc.     

Fatty food,fatty acids,and microglial priming in the adult and aged hippocampus and amygdala

Short-term (3-day) consumption of a high fat diet (HFD) rich in saturated fats is associated with a neuroinflammatory response and subsequent cognitive impairment in aged, but not young adult, male rats. This exaggerated effect in aged rats could be due to a “primed” microglial phenotype observed in the normal aging process in rodents in which aged microglia display a potentiated response to immune challenge. Here, we investigated the impact of HFD on microglial priming and lipid composition in the hippocampus and amygdala of young and aged rats. Furthermore, we investigated the microglial response to palmitate, the main saturated fatty acid (SFA) found in HFD that is proinflammatory. Our results indicate that HFD increased gene expression of microglial markers of activation indicative of microglial priming, including CD11b, MHCII, CX3CR1, and NLRP3, as well as the pro-inflammatory marker IL-1β in both hippocampus and amygdala-derived microglia. Furthermore, HFD increased the concentration of SFAs and decreased the concentration of polyunsaturated fatty acids (PUFAs) in the hippocampus. We also observed a specific decrease in the anti-inflammatory PUFA docosahexaenoic acid (DHA) in the hippocampus and amygdala of aged rats. In a separate cohort of young and aged animals, isolated microglia from the hippocampus and amygdala exposed to palmitate in vitro induced an inflammatory gene expression profile mimicking the effects of HFD in vivo. These data suggest that palmitate may be a critical nutritional signal from the HFD that is directly involved in hippocampal and amygdalar inflammation. Interestingly, microglial activation markers were increased in response to HFD or palmitate in an age-independent manner, suggesting that HFD sensitivity of microglia, under these experimental conditions, is not the sole mediator of the exaggerated inflammatory response observed in whole tissue extracts from aged HFD-fed rats.