用于嗅觉机理研究的MEMS微探针阵列

微探针芯片是基于微机电系统MEMS体加工技术的研究在体神经电生理或临床药理分析的新兴工具,主要应用动物脑区电生理的在体研究,可有助于在原位水平的网络系统中,了解神经元之间的信息耦合传导机制,探索神经元如何联系成整体来处理和储存信息等.首先分析细胞-微电极界面模型,然后介绍了原位式微探针的设计和工艺制作步骤,采用微探针对大鼠嗅球区附近和海马区的脑电位进行测试和分析,并通过对嗅粘膜施加气体刺激,观察嗅前核区电位响应变化,结果显示10-3M的甲基水杨酸盐对大鼠嗅上皮刺激时,会在嗅前核区产生响应.在此基础上,结合当前的研究工作,说明基于体加工MEMS技术的微探针阵列适合于在体神经元的多位点实时测量,对嗅觉传导机理的研究具有重要作用.     

仿生光学人工鼻及其呼吸气体检测的实验研究

针对呼吸气体诊断的需要，介绍了一种新型的具有仿生特点的光学人工鼻实验系统，用于探索通过呼吸气味诊断疾病的可行性。该系统由气敏光纤传感器阵列和信号处理系统组成，利用光纤传感器易与多种聚合物敏感材料相结合和其对气味的响应快、精度高以及易获得嗅觉动态信息的特点，通过计算化学方法，实现了多种聚合物敏感材料与荧光试剂的定量配制，研究了具有不同选择性的气味光纤传感器阵列。此外，将生物嗅觉机理模型用于人工鼻系统的设计过程，采用模拟生物嗅觉的感受器-嗅球-线性延迟神经网络算法，实现了从光纤传感器阵列的响应数据中同时分离出混合气味的成分和浓度信息。实现结果表明，该光学人工鼻传感器实验系统具有一定的仿生特性和应用前景。     

结合磁共振成像和脑机接口的新型在体生物电子鼻的研究

在体生物电子鼻利用了哺乳动物的嗅觉系统,因此具有很高的灵敏度和特异性,但在嗅球中植入电极的过程中其位置主要根据经验确定,因此成功率并不理想。利用锰离子的钙离子相似性和强顺磁性,在10只大鼠单侧鼻腔中滴入锰离子并给予气味刺激,进行磁共振扫描并标记出大鼠嗅球中的对特定气味的响应区域。在该区域中植入微丝阵列电极,记录电生理信号并就行神经信号解码,实验结果表明,受到该特异性气体刺激后,嗅球神经元的电生理信号中LFP信号β波能量增强,spike信号对该气体刺激也会有响应,并且有些通道的spike发放频率变化与刺激气体的浓度有较好的线性关系。此方法对乙酸异戊酯和正丁酸的检测下限分别为0.033和0.007 2 μM。第一次利用锰离子增强磁共振辅助定位的生物电子鼻,未来在爆炸物搜索、食品安全等方面都有广阔的前景。     

小鼠嗅球中水通道蛋白4表达缺失对抑郁发生的影响

目的:观察水通道蛋白4(AQP4)在小鼠嗅球中的表达情况,探讨AQP4表达缺失对小鼠抑郁发生的影响。方法:应用免疫荧光技术观察小鼠嗅上皮和嗅球中AQP4的表达,采用嗅觉电位(EOG)测定比较小鼠的嗅觉差异,悬尾试验(TST)和喷溅试验(ST)检测AQP4基因敲除(AQP4^-/-)小鼠与野生型(WT)小鼠的抑郁样行为,并分析抑郁行为与嗅觉电位的相关性。结果:免疫荧光检测发现AQP4在嗅上皮和嗅球神经纤维层和突触小球层均有不同程度表达; AQP4^-/-与WT小鼠相比,食物性嗅觉电位诱发实验的电位更低(P <0.01);抑郁行为学发现AQP4^-/-小鼠悬尾实验的不动时间较长、第一次梳理背部毛发潜伏时间较长以及梳理时间较短(P <0.01); AQP4^-/-小鼠嗅觉电位与悬尾不动时间、梳理潜伏时间呈负相关(P <0.05),与梳理背部毛发时间呈正相关性(P <0.05)。结论:AQP4表达在小鼠嗅鞘细胞膜上,其表达对于维持嗅觉信号的传递具有重要意义,AQP4基因敲除引起小鼠嗅觉的减退...     

僧帽细胞电位发放的模型分析

本文构造从感觉神经元、僧帽细胞到大脑皮层神经元,并反馈到颗粒细胞的嗅觉神经系统模型.数值分析模型结构中各个神经元的电位发放,特别是嗅小球内的细胞电位变化,结果显示僧帽细胞的发放对感觉神经元的刺激变化较大,僧帽细胞对皮层细胞的相图出现各种模式,从而模型刻画了嗅觉系统中僧帽细胞的信息传递特性.     

早期嗅觉剥夺对SD鼠发育的影响

目的:为了揭示嗅觉发生及其对发育的影响,进行了早期嗅觉剥夺对SD鼠发育影响的研究.方法: 本实验以出生3.5d SD乳鼠应用三氯甲烷滴注法定量定点破坏嗅黏膜,采用神经行为学方法评估其与正常对照组SD鼠的行为学差异,并进一步结合形态学研究揭示嗅脑发育及嗅觉发生的变化.结果: 3.5d乳鼠的早期嗅觉剥夺使乳鼠发育明显迟缓,不能趋利避害;嗅小球消失.结论: 嗅觉及嗅脑对个体整体发育具有重要的调节作用,早期嗅觉剥夺能够使嗅脑结构发生变化,改变嗅脑脑区神经元的突触连接方式.     

嗅成鞘细胞在新生大鼠嗅球的分布

中枢神经系统(CNS)内的胶质微环境是导致再生失败的重要因素。嗅觉神经元(olfactory sensory neurons,OSNs)具有终生再生的能力,因此嗅觉系统成为研究神经发生和轴突生长迁移的良好模型。嗅成鞘细胞(olfactory ensheathing cells,OECs)是决定OSNs轴突能够终生再生的关键因素。周长满等对成年大鼠嗅成鞘细胞的分布进行了研究。     

嗅鞘细胞与神经细胞体外共培养的研究进展

嗅鞘细胞(olfactory ensheathing cells,OECs)分布于嗅球和嗅上皮基底膜,同时具有中枢神经系统星形胶质细胞和周围神经系统雪旺细胞特性,其生物学功能极为广泛.近年研究证实OECs具有支持和促进多种神经元存活和轴突再生的作用,目前已被广泛应用于中枢神经系统损伤的修复[1,2].     

C1型尼曼-匹克症小鼠嗅球细胞的病理变化

目的通过观察NPC1基因突变型小鼠嗅球的形态学变化,探讨C1型尼曼-匹克症对嗅觉系统的影响。方法提取小鼠尾部组织DNA进行聚合酶链反应检测种系基因型;选取出生后第30天的野生型和突变型小鼠,采用免疫组织化学方法观察对比嗅球系统中神经元轴突的形态结构和髓鞘形成,以及神经丝蛋白(SMI31)和髓鞘碱性蛋白(MBP)的表达情况;TUNEL方法检测嗅球神经细胞的凋亡情况。结果 NPC1基因突变型小鼠嗅球中NPC1蛋白表达明显降低,神经细胞凋亡增加;SMI31免疫荧光显示嗅球内SMI31发生变性聚集,导致神经纤维缠结;同时,MBP蛋白表达量明显降低,髓鞘形成受到抑制。结论 NPC1基因突变能够引起嗅球神经细胞发生病理性变化,影响嗅觉系统的正常功能。     

嗅鞘细胞培养上清液对体外培养脊髓神经元存活及活性的影响

目的:通过检测鞘嗅细胞培养上清液,研究其是否能够分泌促进神经元存活及活性的物质,以便为CNS神经损伤修复提供相关移植的理论依据。方法:分离培养嗅神经及嗅球颗粒层(ONGL)细胞,经过传代培养进行纯化嗅鞘细胞,最后收集其培养上清液。浓缩并检测其蛋白浓度,分别以10ug/ml、20ug/ml、40ug/ml、80ug/ml、160ug/ml的浓度加到培养的脊髓神经元的存活及活性指标。结果:嗅鞘细胞培养上清的蛋白浓度为40ug/ml、80ug/ml时对神经元夏活具有促进作用(与对照组相比P<0.01),而80ug/ml时对神经元活性具有促进作用(P<0.01)。结论:嗅鞘细胞在培养状态能够分泌一些活性物质,这些活性物质对神经元的存活和活性有明显的促进作用。     

Nicotine-induced enhancement of glutamatergic and GABAergic synaptic transmission in the mouse amygdala.

Presynaptic nicotinic acetylcholine receptors (nAChRs) are thought to mediate some of the cognitive and behavioral effects of nicotine. The olfactory projection to the amygdala, and intra-amygdaloid projections, are limbic relays involved in behavioral reinforcement, a property influenced by nicotine. Co-cultures consisting of murine olfactory bulb (OB) explants and dispersed amygdala neurons were developed to reconstruct this pathway in vitro. Whole cell patch-clamp recordings were obtained from amygdala neurons contacted by OB explant neurites, and spontaneous and evoked synaptic currents were characterized. The majority of the 108 innervated amygdala neurons exhibited glutamatergic spontaneous postsynaptic currents (PSCs), 20% exhibited GABAergic spontaneous PSCs, and 17% exhibited both. Direct extracellular stimulation of OB explants elicited glutamatergic synaptic currents in amygdala neurons. Antibodies to nAChR subunits co-localized with an antibody to synapsin I, a presynaptic marker, along OB explant processes, consistent with the targeting of nAChR protein to presynaptic sites of the mitral cell projections. Hence, we examined the role of presynaptic nAChRs in modulating synaptic transmission in the OB-amygdala co-cultures. Focal application of 500 nM to 1 microM nicotine for 5-60 s markedly increased the frequency of spontaneous PSCs, without a change in the amplitude, in 39% of neurons that exhibited glutamatergic spontaneous PSCs (average peak fold increase = 125.2 +/- 33.3). Nicotine also enhanced evoked glutamatergic currents elicited by direct stimulation of OB explant fibers. Nicotine increased the frequency of spontaneous PSCs, without a change in the amplitude, in 35% of neurons that exhibited GABAergic spontaneous PSCs (average peak fold increase = 63.9 +/- 34.3). Thus activation of presynaptic nAChRs can modulate glutamatergic as well as GABAergic synaptic transmission in the amygdala. These results suggest that behaviors mediated by olfactory projections may be modulated by presynaptic nAChRs in the amygdala, where integration of olfactory and pheromonal input is thought to occur.     

Expression of transient receptor potential (TRP) channel mRNAs in the mouse olfactory bulb

Transient receptor potential (TRP) channels are a large family of cation channels. The 28 TRP channel subtypes in rodent are divided into 6 subfamilies: TRPC1-7, TRPV1-6, TRPM1-8, TRPP2/3/5, TRPML1-3 and TRPA1. TRP channels are involved in peripheral olfactory transduction. Several TRPC channels are expressed in unidentified neurons in the main olfactory bulb (OB), but the expression of most TRP channels in the OB has not been investigated. The present study employed RT-PCR as an initial survey of the expression of TRP channel mRNAs in the mouse OB and in 3 cell types: external tufted, mitral and granule cells. All TRP channel mRNAs except TRPV5 were detected in OB tissue. Single cell RT-PCR revealed that external tufted, mitral and granule cell populations expressed in aggregate 14 TRP channel mRNAs encompassing members of all 6 subfamilies. These different OB neuron populations expressed 7–12 channel mRNAs. Common channel expression was more similar among external tufted and mitral cells than among these cells and granule cells. These results indicate that a large number of TRP channel subtypes are expressed in OB neurons, providing the molecular bases for these channels to regulate OB neuron activity and central olfactory processing.     

Conditional ablation of Tbr2 results in abnormal development of the olfactory bulbs and subventricular zone‐rostral migratory stream

Development of the olfactory bulb (OB) is a complex process that requires contributions from several progenitor cell niches to generate neuronal diversity. Previous studies showed that Tbr2 is expressed during the generation of glutamatergic OB neurons in rodents. However, relatively little is known about the role of Tbr2 in the developing OB or in the subventricular zone‐rostral migratory stream (SVZ‐RMS) germinal niche that gives rise to many OB neurons. Results: Here, we use conditional gene ablation strategies to knockout Tbr2 during embryonic mouse olfactory bulb morphogenesis, as well as during perinatal and adult neurogenesis from the SVZ‐RMS niche, and describe the resulting phenotypes. We find that Tbr2 is important for the generation of mitral cells in the OB, and that the olfactory bulbs themselves are hypoplastic and disorganized in Tbr2 mutant mice. Furthermore, we show that the SVZ‐RMS niche is expanded and disordered following loss of Tbr2, which leads to ectopic accumulation of neuroblasts in the RMS. Lastly, we show that adult glutamatergic neurogenesis from the SVZ is impaired by loss of Tbr2. Conclusions: Tbr2 is essential for proper morphogenesis of the OB and SVZ‐RMS, and is important for the generation of multiple lineages of glutamatergic olfactory bulb neurons. Developmental Dynamics 243:440–450, 2014. © 2013 Wiley Periodicals, Inc.     

Dopamine modulates synaptic transmission between rat olfactory bulb neurons in culture

The glomerular layer of the olfactory bulb (OB) contains synaptic connections between olfactory sensory neurons and OB neurons as well as connections among OB neurons. A subpopulation of external tufted cells and periglomerular cells (juxtaglomerular neurons) expresses dopamine, and recent reports suggest that dopamine can inhibit olfactory sensory neuron activation of OB neurons. In this study, whole cell electrophysiological and primary culture techniques were employed to characterize the neuromodulatory properties of dopamine on glutamatergic transmission between rat OB mitral/tufted (M/T) cells and interneurons. Immunocytochemical analysis confirmed the expression of tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, in a subpopulation of cultured neurons. D2 receptor immunoreactivity was also observed in cultured M/T cells. Dopamine reduced spontaneous excitatory synaptic events recorded in interneurons. Although the D1 receptor agonist SKF38393 and the D2 receptor agonist bromocriptine mesylate mimicked this effect, evoked excitatory postsynaptic potentials (EPSPs) recorded from monosynaptically coupled neuron pairs were attenuated by dopamine and bromocriptine but not by SKF38393. Neither glutamate-evoked currents nor the membrane resistance of the postsynaptic interneuron were affected by dopamine. However, evoked calcium channel currents in the presynaptic M/T cell were diminished during the application of either dopamine or bromocriptine, but not SKF38393. Dopamine suppressed calcium channel currents even after nifedipine blockade of L-type channels, suggesting that inhibition of the dihydropyridine-resistant high-voltage activated calcium channels implicated in transmitter release may mediate dopamine's effects on spontaneous and evoked synaptic transmission. Together, these data suggest that dopamine inhibits excitatory neurotransmission between M/T cells and interneurons via a presynaptic mechanism.     

切除嗅球对成年大鼠嘴侧迁移流的影响

我们以前的研究观察到嗅球切除后室管膜下层(SVZ)仍能产生新细胞,但新细胞迁移的通路尚不清楚。为此,本研究建立了成年SD雄性大鼠右侧嗅球切除模型,利用Nissl染色、PSA-NCAM和GFAP免疫组织化学染色的方法观察了成年SD大鼠嗅球切除后存活不同时间两侧嘴侧迁移流(RMS)的形态学特征及RMS细胞的密度和单个细胞的面积,并进行统计学分析;同时利用Westernblot方法检测PSA-NCAM在RMS的表达。结果观察到:(1)嗅球切除后不同时间点,嗅球切除侧RMS的细胞数和面积增加,PSA-NCAM免疫阳性细胞数增加,而GFAP免疫阳性细胞数在嗅球切除后2周和4周有明显增加;(2)嗅球切除后两侧RMS的细胞密度和单个细胞的面积没有明显改变;(3)从矢状切片可见嗅球切除后RMS的形态和路径没有明显改变,但在切除断端细胞堆积明显。这些结果提示嗅球切除后仍有年轻神经元沿RMS向嘴侧迁移,SVZ的神经生发活动与嗅球的存在与否可能没有必然的联系。     

Afferents to the rostral olfactory bulb in sheep with special emphasis on the cholinergic, noradrenergic and serotonergic connections

The olfactory bulb (OB) is involved in the processing of olfactory information particularly through the activation of its afferents. To localize their cell origin in sheep, a specific retrograde fluorescent tracer, Fluoro-Gold, was injected into the olfactory bulb of seven ewes. By using immunocytochemical techniques, retrogradely labeled neurons were colocalized with choline acetyltransferase, tyrosine hydroxylase, dopamine-β-hydroxylase and serotonin to characterize cholinergic, noradrenergic and serotonergic Fluoro-Gold-labeled neurons. Most afferents originated from the ipsilateral side of the injection site. The OB received major inputs from the anterior olfactory nucleus (AON), the piriform cortex (PC), the olfactory tubercle, the diagonal band of Broca (DBB) and the amygdala. Other retrogradely labeled neurons were observed in the taenia tecta, the septum, the nucleus of the lateral olfactory tract, the preoptic area, the lateral hypothalamic area, the mediobasal hypothalamus, the lateral part of the premammillary nucleus, the paraventricular nucleus of the hypothalamus, the paraventricular thalamic nucleus, the central grey, the substantia nigra (SN), the ventral tegmental area (VTA), the lateral nucleus to the interpeduncular nucleus (lIP), the raphe and the locus coeruleus (LC). Contralateral labeling was also found in the AON, the PC, the SN compacta, the VTA, the lIP and the LC. Cholinergic Fluoro-Gold-labeled neurons belonged to the horizontal and vertical branch of the DBB. Noradrenergic afferents came from the LC and serotoninergic afferents came from the medial raphe nuclei and the lIP. These data are discussed in relation with olfactory learning in the context of maternal behavior in sheep.     

Olfactory epithelia differentially express neuronal markers

All three olfactory epithelia, the olfactory epithelium proper (OE), the septal organ of Masera (SO), and the vomeronasal organ of Jacobson (VNO) originate from the olfactory placode. Here, their diverse neurochemical phenotypes were analyzed using the immunohistochemical expression pattern of different neuronal markers. The olfactory bulb (OB) served as neuronal control. Neuronal Nuclei Marker (NeuN) is neither expressed in sensory neurons in any of the three olfactory epithelia, nor in relay neurons (mitral/tufted cells) of the OB. However, OB interneurons (periglomerular/granule cells) labeled, as did supranuclear structures of VNO supporting cells and VNO glands. Protein Gene Product 9.5 (PGP9.5 = C-terminal ubiquitin hydrolase L1 = UCHL1) expression is exactly the opposite: all olfactory sensory neurons express PGP9.5 as do OB mitral/tufted cells but not interneurons. Neuron Specific Enolase (NSE) expression is highest in the most apically located OE and SO sensory neurons and patchy in VNO. In contrast, the cytoplasm of the most basally located neurons of OE and SO immunoreacted for Growth Associated Protein 43 (GAP-43/B50). In VNO neurons GAP-43 labeling is also nuclear. In the cytoplasm, Olfactory Marker Protein (OMP) is most intensely expressed in SO, followed by OE and least in VNO neurons; further, OMP is also expressed in the nucleus of basally located VNO neurons. OB mitral/tufted cells express OMP at low levels. Neurons closer to respiratory epithelium often expressed a higher level of neuronal markers, suggesting a role of those markers for neuronal protection against take-over. Within the VNO the neurons show clear apical–basal expression diversity, as they do for factors of the signal transduction cascade. Overall, expression patterns of the investigated neuronal markers suggest that OE and SO are more similar to each other than to VNO.     

Roles of planar cell polarity signaling in maturation of neuronal precursor cells in the postnatal mouse olfactory bulb

Neuronal precursor cells generated by stem cells in the subventricular zone (SVZ) migrate and differentiate into mature interneurons in the olfactory bulb (OB). The mechanisms responsible for the dynamic morphological changes in cells during this process are largely unknown. Wnt/planar cell polarity (PCP) signaling regulates various developmental events, including neuronal migration and neurite formation. Here, we studied the function of two components of the PCP pathway, Dishevelled2 and Van Gogh like-2, in the newborn neurons in the postnatal mouse OB. Electroporation- or lentivirus-mediated introduction of vectors carrying a knockdown or dominant-negative construct of these genes into the SVZ altered the distribution and dendrite formation of newborn neurons in the OB, suggesting that PCP signaling is involved in regulating the maturation of new neurons in the OB.     

Trial-by-trial discrimination of three enantiomer pairs by neural ensembles in mammalian olfactory bulb

Populations of output neurons in the mammalian olfactory bulb (OB) exhibit distinct, widespread spatial and temporal activation patterns when stimulated with odorants. However, questions remain as to how ensembles of mitral/tufted (M/T) neurons in the mammalian OB represent odorant information. In this report, the single-trial encoding limits of random ensembles of putative single- and multiunit M/T cells in the anesthetized rat OB during presentations of enantiomers of limonene, carvone, and 2-butanol are investigated using simultaneous multielectrode recording techniques. The results of these experiments are: the individual constituents of our recorded ensembles broadly represent information about the presented odorants, the ensemble single-trial response of small spatially distributed populations of M/T neurons can readily discriminate between six different odorants, and the most consistent odorant discrimination is attained when the ensemble consists of all available units and their responses are integrated over an entire breathing cycle. These results suggest that small differences in spike counts among the ensemble members become significant when taken within the context of the entire ensemble. This may explain how ensembles of broadly tuned OB neurons contribute to olfactory perception and may explain how small numbers of individual units receiving input from distinct olfactory receptor neurons can be combined to form a robust representation of odorants.