Identification and characterization of an output neuron from the oscillatory molluscan olfactory network.

Synchronous oscillations in olfactory systems have been thought to play critical roles in encoding olfactory information. However, their role in determining behavior is unknown. As a first step toward understanding the decoding process of coherent oscillation, we looked for a neuron in the terrestrial slug Limax marginatus that receives output signals from the procerebrum (PC), which is the olfactory center of Limax. We identified a neuron in the metacerebrum that extends its neurites into both the PC and the metacerebrum, and named it the metacerebro-procerebral neuron (MPN). The MPN exhibited a membrane potential oscillation that was synchronous with the local field potential oscillation in the PC. When we cut the PC off, the membrane potential oscillation of the MPN disappeared. Numerous varicosities were found on the neurites in the metacerebrum, while no varicosities were found on the neurites inside the PC. From these morphological and physiological results, we conclude that the MPN is an output neuron from the PC. The MPN also receives monosynaptic inputs from the superior and inferior tentacle nerves. The MPN thus may receive olfactory information from two pathways, one directly from the sensory organ and the other by way of the PC, possibly functioning to integrate them.     

Molecular cloning and expression of the rat monocyte chemotactic protein-3 gene: a possible role in stroke.

Using the suppression subtractive hybridization (SSH) strategy for differential gene cloning, we identified the induced expression of a rat homologue to murine and human monocyte chemotactic protein-3 (MCP-3) in ischemic brain. The 2.4-kilobase rat MCP-3 gene features high homology in gene structure and sequence to murine MCP-3. The temporal expression of MCP-3 mRNA was examined in brain tissue rendered ischemia by permanent or temporary occlusion of the middle cerebral artery (MCAO). A marked increase in MCP-3 mRNA was observed 12 h post-ischemia, with 49-fold and 17-fold increase (n=4, p<0.01) over control in the permanent or temporary MCAO, respectively. Significant induction of MCP-3 in the ischemic cortex was sustained up to 5 days after ischemic injury. The profile of MCP-3 mRNA induction paralleled leukocyte infiltration and accumulation that occur after focal stroke, suggesting a role for MCP-3 in recruiting these inflammatory cells into the ischemic tissue. Molecular cloning of rat MCP-3 should provide a valuable tool, as demonstrated in the present work, for the investigation of MCP-3 expression and function in rat disease models.     

Dysregulation of olfactory receptor neuron lineage in schizophrenia.

BACKGROUND: Growing evidence implicates abnormal neurodevelopment in schizophrenia. While neuron birth and differentiation is largely completed by the end of gestation, the olfactory epithelium (OE) is a unique part of the central nervous system that undergoes regeneration throughout life, thus offering an opportunity to investigate cellular and molecular events of neurogenesis and development postmortem. We hypothesized that OE neurons exhibit deviant progress through neurodevelopment in schizophrenia characterized by an increase in immature neurons. METHODS: Olfactory epithelium was removed at autopsy from 13 prospectively assessed elderly subjects who had schizophrenia and 10 nonpsychiatric control subjects. Sections were immunolabeled with antibodies that distinguish OE neurons in different stages of development, including basal cells (low-affinity nerve growth factor receptor, p75NGFR), postmitotic immature neurons (growth-associated protein 43 [GAP43]), and mature olfactory receptor neurons (olfactory marker protein). Absolute and relative densities of each cell type were determined. RESULTS: We observed a significantly lower density of p75NGFR basal cells (37%) in schizophrenia and increases in GAP43 + postmitotic immature neurons (316%) and ratios of GAP43 + postmitotic immature neurons to p75NGFR + cells (665%) and olfactory marker protein + mature neurons to p75NGFR + basal cells (328%). Neuroleptic-free schizophrenia subjects exhibited the highest GAP43 + postmitotic immature neuron values. CONCLUSIONS: Abnormal densities and ratios of OE neurons at different stages of development indicate dysregulation of OE neuronal lineage in schizophrenia. This could be because of intrinsic factors controlling differentiation or an inability to gain trophic support from axonal targets in the olfactory bulb. While caution is necessary in extrapolating developmental findings in mature OE to early brain development, similarities in molecular events suggest that such studies may be instructive.     

Projections from orbitofrontal cortex to anterior piriform cortex in the rat suggest a role in olfactory information processing

The orbitofrontal cortex (OFC) has been characterized as a higher-order, multimodal sensory cortex. Evidence from electrophysiological and behavioral studies in the rat has suggested that OFC plays a role in modulating olfactory guided behavior, and a significant projection to OFC arises from piriform cortex, the traditional primary olfactory cortex. To discern how OFC interacts with primary olfactory structures, the anterograde tracer Phaseolus vulgaris leucoagglutinin was injected into orbitofrontal cortical areas in adult male rats. Labeled fibers were found in the piriform cortex and olfactory bulb on the side ipsilateral to the injection. Notably, the projection to piriform cortex was predominantly from ventrolateral orbital cortex, and was not uniform; rostrally, the projection to the ventral portion of the anterior piriform cortex (APC) was substantial, while the dorsal APC was virtually free of labeled fibers. Labeled fibers were found in both the dorsal and ventral portions in more caudal regions of APC. Most labeled fibers were found in layer III, although a substantial number of fibers were observed in layers Ib and II. Labeled fibers in posterior piriform cortex also were seen after injection into orbitofrontal areas. Taken together with previous reports, these findings suggest that piriform cortex includes multiple subdivisions, which may perform separate, parallel functions in olfactory information processing. Further, these results suggest that the OFC, in addition to its putative role in encoding information about the significance of olfactory stimuli, may play a role in modulating odor response properties of neurons in piriform cortex.     

Neurturin基因克隆及其在Vero细胞中的表达

BACKGROUND： Transplantation of cell lines expressing neurturin （NTN） has been used to treat animal models of Parkinson＇s disease. However, gone homology between humans and rats varies greatly, so experimental results are not entirely suitable for understanding cellular transplantation in humans. OBJECTIVE： To explore expression of NTN in African green monkey kidney cells （Vero cells）; to obtain a stably NTN expressing cell line. DESIGN, TIME AND SETTING： An observation study of gone engineering and cellular biology was performed at the Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College between 2005 and 2008. MATERIALS： Human embryonic hepatic tissues, expression vector pcDNA3, and Vero cells were prepared in this laboratory. Primers were synthesized by TaKaRa Biotechnology, Dalian, China; RNA extraction kit and plasmid extraction kit were purchased from Shanghai Watson Bioengineering, China; G418 and MTT were purchased through Sigma, USA; Lipofectamine2000 was a product of Invitrogen, USA; mice anti-human NTN antibody and fluorescent labeling goat anti-mouse IgG antibody were provided by Jingmei Biotech, China. METHODS： Total RNA was harvested from human embryonic hepatic tissues, and NTN cDNA was cloned by RT-PCR method, followed by subcloning into the pcDNA3 eukaryotic expression vector. The obtained pcDNA3/hNTN was stably transfected into Vero cells using Lipofectamine 2000, and stably expressing clones were selected using G418. MAIN OUTCOME MEASURES： NTN mRNA and protein expressions were respectively identified by RT-PCR and immunofluorescence. The morphology of transfected cells was observed under inverted microscopy, and the growth characteristics of those cells were determined using MTT method. RESULTS： A clonal cell line, stably expressing human NTN mRNA and protein, was obtained through stable transfection of pcDNA3/hNTN into Vero cells. The transfected Veto cells exhibited irregular morphology, rather than a spindle shape. The growth retardation phase was prolonged, but the number of cells was identical to non-transfected cells. CONCLUSION： Vero cell lines, which stably expressed human NTN protein, were obtained, and expression patterns of these cell lines were acceptable.     

Hormonal control of neuropeptide gene expression in sexually dimorphic olfactory pathways.

An abundance of experimental literature has established that gonadal steroid hormones are responsible for the sexual differentiation of neural circuitry, mediating a variety of reproductive behaviors and physiological mechanisms. These same hormones regulate the expression of reproductive function in the adult and may influence the responsiveness of the brain to specific olfactory cues. The recent demonstration that the expression of the neuropeptide cholecystokinin is activationally regulated by estrogen at the mRNA level, within a sexually dimorphic population of neurons in the medial amygdala, suggests a possible cellular mechanism for the hormonal modulation of olfactory information relayed along the vomeronasal pathway to the hypothalamus.     

恶性星形胶质瘤中转录因子的表达异常

目的研究人类星形胶质瘤中转录因子(TF)的表达异常.方法用cDNA微阵列方法研究人类正常大脑和星形胶质瘤中转录因子的表达.共3个标本标本1混合了3例三级星形胶质瘤,标本2混合了2例四级星形胶质瘤(胶质母细胞瘤,GBM),标本3为对照,混合了4例正常大脑组织.33p标记的cDNA与带有14 000个cDNA片断的微阵列膜杂交,通过比较肿瘤标本和对照标本的光密度来分析各基因,相差2倍以上被认为表达有差异.结果普查了109个转录因子.GBM标本中转录因子的表达高于三级胶质瘤(表达比率为1.33±0.28和1.10±0.16,P＜0.001).在2个肿瘤标本中共有13个异常表达的转录因子.在三级胶质瘤中有4个上调基因和1个下调基因,在GBM中有6个上调基因和5个下调基因.转录因子ⅡB在2个标本中均下调(三级胶质瘤中为0.21倍,GBM中为0.28倍).有2个因子在2个标本中均上调,它们为HMGI-C(分别为2.17倍和4.13倍),和基本转录因子2(BTF2)63kDa亚单位(分别为2.81倍和2.69倍).结论在三级胶质瘤和GBM中有转录因子的异常表达.TFⅡB、HMGI-C、BTF2 63kDa亚单位是进一步研究的目标.     

Neuronal expression and interaction with the synaptic protein CASK suggest a role for Neph1 and Neph2 in synaptogenesis

Formation, differentiation, and plasticity of synapses require interactions between pre- and postsynaptic partners. Recently, it was shown that the transmembrane immunoglobulin superfamily protein SYG-1 is required for providing synaptic specificity in C. elegans. However, it is unclear whether the mammalian orthologs of SYG-1 are also involved in local cell interactions to determine specificity during synapse formation. We used in situ hybridization, immunohistochemistry, and immunogold electron microscopy to study the temporal and spatial expression of Neph1 and Neph2 in the developing and adult mouse brain. Both proteins show similar patterns with neuronal expression starting around embryonic days 12 and 11, respectively. Expression is strongest in areas of high migratory activity. In the adult brain, Neph1 and Neph2 are predominantly seen in the olfactory nerve layer and the glomerular layer of the olfactory bulb, in the hippocampus, and in Purkinje cells of the cerebellum. At the ultrastructural level, Neph1 and Neph2 are detectable within the dendritic shafts of pyramidal neurons. To a lesser extent, there is also synaptic localization of Neph1 within the stratum pyramidale of the hippocampal CA1 and CA3 region on both pre- and postsynaptic sites. Here it colocalizes with the synaptic scaffolder calmodulin-associated serin/threonin kinase (CASK), and both Neph1 and Neph2 interact with the PDZ domain of CASK via their cytoplasmic tail. Our results show that Neph proteins are expressed in the developing nervous system of mammals and suggest that these proteins may have a conserved function in synapse formation or neurogenesis.     

Region-specific consequences of PCD gene expression in the olfactory system

These studies investigated the response of olfactory bulb juxtaglomerular dopamine neurons to the loss of mitral cells in 6-7-month-old Purkinje cell degeneration (PCD) mice. Previous studies in normal mice, with tyrosine hydroxylase (TH) enzyme as a marker, demonstrated that following peripheral olfactory afferent denervation the juxtaglomerular dopamine neurons exhibited a large reduction in TH activity and immunoreactivity. These intrinsic dopamine neurons also receive afferent input via dendrodendritic contacts with mitral cells. In contrast to the deficits produced by peripheral denervation, following mitral cell degeneration in homozygous recessive PCD mice, TH activity and immunoreactivity were unaltered as compared to normal heterozygous littermates. Moreover, TH activity in the substantia nigra also was unchanged, thus suggesting that the dopamine phenotype is resistant to the influences of the pcd gene. Despite the absence of a well-defined effect of the pcd gene on neurons bearing the TH phenotype, the expression of this mutation within the olfactory system is not limited to mitral cell degeneration. The current studies also demonstrate the absence of the anterior commissure, especially pars anterior, in homozygous recessive PCD mice at 6-7 months postnatal. Whether or not the loss of the anterior commissure is a primary effect or one that is secondary to mitral cell degeneration, this structural alteration provides evidence that the pcd gene exerts more widespread effects within the olfactory system that previously appreciated. The neuronal specificity of those effects remains apparent as indicated by the lack of change in TH expression.     

Slow changes of tyrosine hydroxylase gene expression in dopaminergic brain neurons after neurotoxin lesioning: a model for neuron aging.

Slow neuron regression develops during the adult phase of life in select brain systems of mammals. We describe a model in adult rats that resolves several phases in a slow atrophic process that differentially influences levels of mRNA and protein for tyrosine hydroxylase (TH). Responses of striatal dopaminergic markers to 6-hydroxydopamine (6-OHDA) lesions in rats indicated that the striatal terminals maintained TH protein, despite greater than 3-fold loss of TH mRNA in the substantia nigra pars compacta (SNC) cell bodies whose axons project to the striatum. The loss of TH mRNA/cell was progressive up to 9 months, whereas SNC cell body shrinkage stabilized by 3 months post-lesioning. Consideration of possible mechanisms in protein turnover motivated a search for PEST motifs in the TH of rats and other vertebrates that could be a point of regulation by altering the rate of TH protein turnover.     

Early neuron differentiation in the mouse of olfactory bulb. I. Light microscopy

The relation between the growth of axons and the development of their myelin sheaths was determined in rats for normal, myelinating sciatic nerves, and for an experimental model allowing to retard or accelerate axon growth. Axon caliber was measured, and sheath development was determined from measurements of thickness (light microscopy), from counts of the number of turns of myelin lamellae (electron micrographs), and from the rate of the incorporation of acetate-H³ into ether-ethanol extractable lipids. An excellent correlation between changes in sheath thickness and acetate-H³ incorporation was obtained for all experiments. For the myelinating nerves the changes in the rate of acetate-H³ incorporation were related specifically to the rate with which new length of myelin leaflet was added to the existing turns of sheath. The rate of axonal growth was manipulated by applying a snug ligature around the nerve by the fourteenth day, allowing the nerve to compress itself by its own growth. Most axons passed the constriction without interruption, but they were markedly hypoplastic distal to the constriction. After removal of the ligature these axons regrew to their normal caliber ranges. Examination of myelin sheath development in this model showed that retardation of axon growth retarded sheath growth, while acceleration of axon growth accelerated sheath growth. Thus, the rate of axon growth appeared to be the factor controlling the rate of myelin formation by the sheath cells. An appendix describes a model consisting of two interrelated feedback mechanisms by which expansion of the axon may directly control the number of turns of myelin lamellae formed by the sheath cell. The model correspons on all points to established features in the fine structure of fibers.     

Correlated patterns of neuron differentiation and Hox gene expression in the hindbrain: a comparative analysis

Hindbrain neurons are organized into coherent subpopulations with characteristic projection patterns and functions. Many of these serve vital functions that have been conserved throughout the vertebrate radiation, but diversification to modified or highly specialized functions has also occurred. The differentiation of identifiable neuron groups in specific spatial domains must involve the regional expression of determinants within the hindbrain neuroepithelium. The Hox genes are involved in longitudinal regionalization of the neural tube, and their expression patterns in the hindbrain are closely related to the rhombomeres which partition the hindbrain into morphogenetic units. Hox gene expression also exhibits conserved patterning as well as phylogenetic variation. One plausible mechanism that may have contributed to evolutionary diversification in hindbrain neuron populations is therefore the emergence of species-specific differences in Hox gene expression. This article presents a comparative overview of the regional patterning of selected Hox genes and hindbrain neuron populations in several embryologically important species. Although tantalizing correlations exist, the relationship between Hox genes and neuronal patterning is complex, and complicated by dynamic features in each. Much more comparative and developmental data must be obtained before the link between Hox gene expression and hindbrain neuron patterning can be elucidated satisfactorily in an evolutionary context.