Proteome profile of the mature rat olfactory bulb

Olfactory bulbs (OBs) are one of the few brain areas, which show active neurogenesis and neuronal migration processes in adult rats. We constructed a proteome map of the 21 days old rat OBs and identified total 196 proteins, out of which 76 proteins were not reported earlier from rat brain. This includes 24 neuronal activity‐specific proteins present at high levels, 7 of which are reported for the first time from OBs.     

Calbindin D-28k-positive neurons in the rat olfactory bulb

Summary We have studied the distribution of calbindin D-28k immunoreactivity in the rat olfactory bulb using specific monoclonal antibodies and the avidin-biotin-immunoperoxidase method. The largest number of positive neurons was located in the periglomerular layer. These neurons were identified as periglomerular cells; they have been described also by other authors as calbindin-positive elements. Close to these neurons, a second population of nerve cells was identified as superficial shortaxon neurons. The remaining layers showed a smaller number of stained elements. Other labeled neurons were located along the external border of the external plexiform layer; the scarce neurons marking its internal border were identified as van Gehuchten cells. No immunoreactive structures were found in the mitral cell layer, although we observed another population of immunostained short-axon cells at its internal border. Some reactive structures, identified by us as horizontal and vertical cells of Cajal, were located in the boundary zone between the internal plexiform layer and the granule layer. In the white matter, we found a neuronal type characterized by its large size and oriented arborization of varicose dendrites.     

Establishment of primary cultures of rat olfactory bulb under serum-free conditions for studies of cellular injury

Olfactory dysfunction has been implicated in various neurodegenerative diseases including Parkinson's and Alzheimer's disease but, despite intense interest in the neurobiology of the olfactory bulb (OB), studies of neurodegenerative mechanisms have not been attempted in primary OB cultures. This study was aimed at developing a primary OB culture under serum-free conditions in order to investigate injury and excitotoxicity in vitro. Olfactory bulbs from rat pups were rapidly trypsinised and mechanically dissociated and the resultant single cell suspension was centrifuged through a high bovine serum albumin concentration gradient to reduce cellular debris before being seeded in multi-well culture plates. Cells were plated in neurobasal medium containing 0.5 mM glutamine, 25 mM K⁺, 2% B27 and 10% fetal calf serum (FCS) for 24 h and, after 1 day in vitro (div1), were maintained without FCS. At div8, neurones exhibited extensive neuritic networks, were present as a monolayer and were mainly bipolar and immunopositive for γ-aminobutyric acid indicating that they were intrinsic OB neurones. At div8, neurones (positive for microtubule-associated protein-2, 73%) predominated over astrocytes (positive for glial fibrillary acidic protein, 27%). Cellular injury produced by staurosporine, hydrogen peroxide and kainate, when assessed by morphological and biochemical procedures, was shown to be concentration-dependent and significantly reduced the numbers of neurones and astrocytes. Further analyses of kainate-induced injury revealed the presence of TUNEL-positive cells (indicative of apoptosis) and increases in intracellular free calcium, both of which were antagonised by CNQX. Thus, the serum-free culture developed here is amenable to morphological and high throughput neurochemical analyses of mechanisms contributing to the injury of OB neurones in vitro. This work was supported by a Program Grant (no. 236805) from NH&MRC (Australia), of which P.M.B. is a Research Fellow.     

Expression of EGR-1 in a subset of olfactory bulb dopaminergic cells

In the adrenal medulla, binding of the immediate early gene (IEG) proteins, EGR-1 (ZIF-268/KROX-24/NGFI-A) and AP-1, to the tyrosine hydroxylase (Th) proximal promoter mediate inducible Th expression. The current study investigated the potential role of EGR-1 in inducible Th expression in the olfactory bulb (OB) since IEGs bound to the AP-1 site in the Th proximal promoter are also necessary for activity-dependent OB TH expression. Immunohistochemical analysis of a naris-occluded mouse model of odor deprivation revealed weak EGR-1 expression levels in the OB glomerular layer that were activity-dependent. Immunofluorescence analysis indicated that a majority of glomerular cells expressing EGR-1 also co-expressed TH, but only small subset of TH-expressing cells contained EGR-1. By contrast, granule cells, which lack TH, exhibited EGR-1 expression levels that were unchanged by naris closure. Together, these finding suggest that EGR-1 mediates activity-dependent TH expression in a subset of OB dopaminergic neurons, and that there is differential regulation of EGR-1 in periglomerular and granule cells.     

达乌尔黄鼠犁鼻器和副嗅球的组织结构及嗅球c-Fos表达的季节变化

啮齿动物的犁鼻器和副嗅球与社会通讯和生殖行为有关,主嗅球影响其觅食行为。达乌尔黄鼠（Spermophilus dauricus）是一种具有较低社会行为的储脂类冬眠动物。本研究用组织学和免疫组织化学方法探究了其犁鼻器和副嗅球的结构特点及嗅球神经元活动对季节变化的适应。结果发现,达乌尔黄鼠犁鼻器具有较大的血管,犁鼻器管腔外侧为非感觉性的呼吸上皮（Respiratory epithelium,RE）,内侧为感觉上皮（Sensory epithelium,SE）,RE较SE薄,靠近管腔处为假复层柱状上皮。选取犁鼻器中间部位比较,发现SE的厚度、长度及感觉细胞密度均无性别差异。副嗅球位于主嗅球后方背内侧,由6层细胞构成。侧嗅束穿过副嗅球,位于颗粒细胞层之上。雄性达乌尔黄鼠较雌性有更长的僧帽细胞层和颗粒细胞层。春季（3月）和冬季（1月）达乌尔黄鼠主嗅球的嗅小球层、僧帽细胞层和颗粒细胞层的c-Fos-ir神经元密度显著低于夏季（7月）和秋季（10月）,且冬季外网织层的c-Fos-ir神经元密度显著低于夏季和秋季,说明达乌尔黄鼠在冬季和春季的嗅觉神经活动较弱,呈现出对冬眠的生理性适应。这些结果丰富了动物犁鼻器和副嗅球的形态学资料,并有助于理解冬眠动物嗅觉系统对季节变化和冬眠的适应。     

Independent development of presynaptic specializations in olfactory cortex of the fetal rat

Summary Electron microscopy was used to study synaptogenesis in prepyriform cortex of fetal rat pups during early stages of synapse formation. Of special interest is the frequent occurrence of unapposed, developing synaptic specializations in axon and growth cone profiles. The location and morphology of the unapposed specializations suggests that thay are presynaptic in nature. These presumably immature presynaptic specializations are found in the lateral olfactory tract and subjacent cortex. Intermediate forms between uncontacted presynaptic specializations and definitive synapses suggest a synaptogenic sequence in which initial development of an immature presynaptic specialization begins without apposition of a postsynaptic element at that location. This implies that initiation of presynaptic development is not dependent upon postsynaptic contact and also raises the question of whether synaptic contacts could be established via presynaptic induction of postsynaptic formation.Dr. Westrum is an Affiliate of the CDMRC at the University of Washington. The research was supported in part by NIH Grants NS09678, NS17111 (NINCDS), and DE04942 (NIDR), DHHS     

Contractility and structure of adult rat seminiferous tubules in organ culture

Summary Isolated pieces of seminiferous tubules of adult rats were grown in organ culture for up to 8 weeks in Petri dishes on the surface of nutrient agar. The medium consisted of newborn calf serum, Eagle's minimum essential medium, glutamate and antibiotics. This method allowed observation of the contractions of the seminiferous tubules in the culture. Contractility, light and electron microscopic structure and histochemically demonstrable activities of alkaline phosphatase and ATPase of the tubule walls were studied at 1-week intervals. The contractility and alkaline phosphatase activity were maintained in the tubule wall for 3 weeks, and the activity of ATPase was maintained for 5 weeks. The thin filaments of the myoid cells, which are responsible for the contractility, were seen with the electron microscope in tubules cultured for 5 weeks. The organ culture method described in the present paper seems to be valuable for studies concerning the functioning of the myoid cells of the seminiferous tubules and the possibility that this is regulated by hormones.     

Spatiotemporal distribution of the insulin-like growth factor receptor in the rat olfactory bulb

Insulin-like growth factor I (IGF-I) and its receptor (IGF-IR) are involved in growth of neurons. In the rat olfactory epithelium, we previously showed IGF-IR immunostaining in subsets of olfactory receptor neurons. We now report that IGF-IR staining was heaviest in the olfactory nerve layer of the rat olfactory bulb at embryonic days 18, and 19 and postnatal day 1, with labeling of protoglomeruli. In the adult, only a few glomeruli were IGF-IR-positive, some of which were unusually small and strongly labeled. Some IGF-IR-positive fibers penetrated deeper into the external plexiform layer, even in adults. In developing tissues, IGF-IR staining co-localized with that for olfactory marker protein and growth associated protein GAP-43, but to a lesser extent with synaptophysin. In the adult, IGF-IR-positive fibers were compartmentalized within glomeruli. IGF-I may play a role in glomerular synaptogenesis and/or plasticity, possibly contributing to development of coding patterns for odor detection or identification.     

Retinal and olfactory bulb precursor cells show distinct responses to FGF-2 and laminin

We analyzed whether the embryonic (E12.5-E14.5) mouse retina possesses genuine neural stem cells and how they respond to defined growth factors and extracellular matrix molecules. Whereas most combinations produced no or limited cell survival and proliferation in culture, FGF-2 plus heparin and laminin stimulated proliferation and the formation of aggregates composed, after two days, of 95.2% nestin-positive cells. However, cells in these aggregates could only be passaged poorly, lost nestin expression and proliferative capacity, and differentiated into neurons. Under the same conditions, olfactory bulb precursor cells divided efficiently and could be expanded. These data suggest that, in addition to FGF-2 and laminin, embryonic retinal neuroepithelial cells need additional extrinsic and/or intrinsic regulators to maintain cell proliferation and self-renewal.     

Gap junctions between astrocytes during growth and differentiation in organ culture systems

Summary Fetal rat neocortex maintained in organ culture systems with the use of sponge foam matrices and millipore filter platforms undergoes growth and cytodifferentiation along classical neuronal and glial lines up to 36 days in vitro (DIV). Astrocytic differentiation is characterized by accumulation of 80–90 Å glial filaments in the cell bodies and processes of astrocytes. Gap or nexus junctions closely resembling those formed in mammalian brain in situ are identified by 15 DIV. By 36 DIV, interastrocytic gap junctions are numerous and frequently join extensive lengths of adjacent glial plasma membranes. The results suggest that these organ culture systems may provide a favorable environment for the study of cellular structure and function of coupled neuroglia.This work was supported by a research grant from the Veterans Administration. Skilled technical assistance was provided by Robin L. Isaacs and Marilyn Woodward     

Electron microscopic study of the giant cells in the olfactory bulb of labyrinth fish (Belontiidae,Perciformes)

Summary The giant cells in the olfactory bulb of labyrinth fish, most likely belonging to the nervus terminalis, show ultrastructural features of active synthesis of secretory material. Dense core vesicles (70–100 nm in diameter), found in the perikaryon as well as in the axon, are the possible storage sites of the secretory substance. Its chemical nature is unknown. In some of these vesicles acid phosphatase is demonstrable. Large membrane-bound bodies (up to 2000 nm in diameter), either containing an electron dense matrix or debris of cytoplasmic organelles, are also acid phosphatase-positive, suggesting their lysosomal nature. Some other ultrastructural characteristics of these cells are also described.     

Neuronal circuits and computations: Pattern decorrelation in the olfactory bulb

Neuronal circuits in the olfactory bulb transform odor-evoked activity patterns across the input channels, the olfactory glomeruli, into distributed activity patterns across the output neurons, the mitral cells. One computation associated with this transformation is a decorrelation of activity patterns representing similar odors. Such a decorrelation has various benefits for the classification and storage of information by associative networks in higher brain areas. Experimental results from adult zebrafish show that pattern decorrelation involves a redistribution of activity across the population of mitral cells. These observations imply that pattern decorrelation cannot be explained by a global scaling mechanism but that it depends on interactions between distinct subsets of neurons in the network. This article reviews insights into the network mechanism underlying pattern decorrelation and discusses recent results that link pattern decorrelation in the olfactory bulb to odor discrimination behavior.     

Immunohistochemical study on adenohypophysial primordia in organ culture

Summary Rathke's pouches isolated from rat fetuses on day 12 were maintained in organ culture for 9 days and investigated immunohistochemically to test whether or not the hypothalamus is involved in the cytodifferentiation of the adenohypophysis. The unlabeled antibody enzyme method demonstrated that the cultured tissue contains different types of glandular cells, i.e., adrenocorticotropin (ACTH)-, growth hormone (GH)-, luteinizing hormone (LH)-, thyrotropin (TSH)-, and prolactin-producing cells. Indirect evidence was also obtained to indicate the presence of melanocyte stimulating hormone (MSH)-cells. These findings suggest that adenohypophysial primordial cells of rats start to synthesize their respective hormones without stimuli from neurosecretory substances of the brain which are known to be essential for the maintenance of the secretory activity of the adult gland.We wish to express our thanks to Dr. A. Kawaoi for providing anti-porcine ^1–39ACTH, to Dr. S.S. Spicer for the supply of anti-porcine ^17–39ACTH and to Dr. P. Petrusz for the gift of antisera against bovine GH, bovine TSH, HCG and rat prolactin. We should also like to thank Mr. Y. Okamura for technical help and Mr. I. Shimada for preparation of the photographs.     

Hippo-YAP/TAZ信号通路在干细胞增殖、分化及器官再生中的研究进展

Hippo-YAP/TAZ信号通路最初在果蝇中被发现,是器官发育和肿瘤生长过程中重要的调节者。通过调控细胞增殖、凋亡和分化等过程影响器官再生。近年来,对于Hippo-YAP/ TAZ信号通路在调节干细胞 (SC)增殖、自我更新及分化过程中的相关机制有了较大进展。本综述拟通过介绍Hippo-YAP/TAZ信号通路在SC增殖及多向分化过程中的作用、调控机制及器官再生方面的研究进展,为应用SC治疗疾病提供相关理论基础。     

刺猬嗅球冬眠期与非冬眠期c-Fos 表达的差异

利用免疫组化法检测c-Fos 蛋白在不同季节刺猬嗅球各层次的表达差异,探讨c-Fos、嗅觉、冬眠三者的关系。分别选取春、夏、秋、冬四个季节各6 只野生健康刺猬,固定剥离嗅球,石蜡切片,免疫组化显色,拍片,载入Motic Images Advanced 3.2 软件,测量四个季节刺猬嗅球各层次c-Fos 的表达率,将结果载入GraphPadPrism4 软件分析,Microsoft Excel 作图。结果表明:c-Fos 蛋白在成年刺猬嗅球各层均有不同程度的表达,阴性对照不着色,且表现出明显的季节性差异。1)与秋季相比,冬眠期c-Fos 蛋白在刺猬嗅球各层次的表达均有极显著的降低(P <0.01);2)与夏季相比,冬眠期c-Fos 蛋白在外网丛层、僧帽细胞层、颗粒细胞层的表达有极显著降低(P < 0. 01),在嗅神经层、嗅小球层、室管膜层的表达也有显著降低(P < 0. 05);3)与冬眠期相比,春季c-Fos 蛋白在嗅小球层、僧帽细胞层、颗粒细胞层的表达有极显著的升高(P <0. 01),在嗅神经层、外网丛层、室管膜层的表达却没有显著变化(P ﹥ 0.05);4)嗅神经层c-Fos 的表达在春季显著低于秋季,夏季与秋季没有显著差异。颗粒细胞层夏季显著低于秋季(P < 0.05)。秋季c-Fos 在其余各层次的表达与春季、夏季相比都有极显著的提高(P <0.01)。结论:秋季刺猬嗅球神经元最活跃,嗅觉最灵敏,冬眠期刺猬嗅球活跃性大大降低,嗅觉系统最迟钝。c-Fos 在刺猬嗅球中的强表达表明其在嗅觉信息的传递中可能发挥一定作用,c-Fos 表达率的显著季节性差异揭示了刺猬嗅球的活跃性与其冬眠具有一定的相关性。     

Renewing olfactory receptor neurons in goldfish do not require contact with the olfactory bulb to develop normal chemical responsiveness

This study investigated whether contact with the olfactory bulb was necessary for developing and renewing olfactory receptor neurons (ORNs) to attain normal odorant responsiveness, and whether the anatomical and functional recoveries of the olfactory epithelium were similar in both bulbectomized (BE) and bilaterally axotomized (AX) preparations. In vivo electrophysiological recordings were obtained in response to amino acids, a bile acid [taurolithocholic acid sulfate(TLCS)] and a pheromonal odorant [17α, 20β,-dihydroxy-4-pregnen-3-one (17,20P)] from sexually immature goldfish. Both transmission and scanning electron microscopy indicated that the olfactory epithelium degenerated in BE and AX goldfish. Within 1–2 weeks subsequent to the respective surgeries, responses to high concentrations (>0.1 mmol · l⁻¹) of the more stimulatory amino acids remained, whereas responses were no longer obtainable to TLCS and 17,20P. At 4 weeks, responses to amino acid stimuli recovered to control levels, while responses to TLCS and 17,20P were minimal. By 7 weeks post bilateral axotomy, the olfactory epithelium recovered to a condition similar to control sensory epithelium; however, the rate of degeneration and proliferation of receptor neurons in BE preparations appeared to remain in balance, thus blocking further recovery of the olfactory epithelium. At 7 weeks post surgery, odorant responses of AX and BE goldfish to TLCS and 17,20P were still recovering. Accepted: 14 June 1997