Vanilloid Receptor-1 (TRPV1) Expression and Function in the Vasculature of the Rat

Transient receptor potential (TRP) cation channels are emerging in vascular biology. In particular, the expression of the capsaicin receptor (TRPV1) was reported in vascular smooth muscle cells. This study characterized the arteriolar TRPV1 function and expression in the rat. TRPV1 mRNA was expressed in various vascular beds. Six commercially available antibodies were tested for TRPV1 specificity. Two of them were specific (immunostaining was abolished by blocking peptides) for neuronal TRPV1 and one recognized vascular TRPV1. TRPV1 was expressed in blood vessels in the skeletal muscle, mesenteric and skin tissues, as well as in the aorta and carotid arteries. TRPV1 expression was found to be regulated at the level of individual blood vessels, where some vessels expressed, while others did not express TRPV1 in the same tissue sections. Capsaicin (a TRPV1 agonist) evoked constrictions in skeletal muscle arteries and in the carotid artery, but had no effect on the femoral and mesenteric arteries or the aorta. In blood vessels, TRPV1 expression was detected in most of the large arteries, but there were striking differences at level of the small arteries. TRPV1 activity was suppressed in some isolated arteries. This tightly regulated expression and function suggests a physiological role for vascular TRPV1.     

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.     

Attractive and permissive activities of semaphorin 5A toward dorsal root ganglion axons in higher vertebrate embryos

Elongation of the efferent fibers of dorsal root ganglion (DRG) neurons toward their peripheral targets occurs during development. Attractive or permissive systems may be involved in this elongation. However, the molecular mechanisms that control it are largely unknown. Here we show that class 5 semaphorin Sema5A had attractive/permissive effects on DRG axons. In mouse embryos, Sema5A was expressed in and around the path of DRG efferent fibers, and cell aggregates secreting Sema5A attracted DRG axons in vitro. We also found that ectopic Sema5A expression in the spinal cord attracted DRG axons. Together, these findings suggest that Sema5A functions as an attractant to elongate DRG fibers and contributes to the formation of the early sensory network.     

Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury.     

美洲大蠊中枢DUM神经元的分离和电压门控Na+电流的记录

【目的】建立美洲大蠊Periplaneta americana中枢神经系统背侧不成对中间神经元（dorsal unpaired median neurons, DUM neurons）的分离方法和DUM神经元电生理实验模型。【方法】IA型胶原酶法消化美洲大蠊末端腹神经节, 机械吹打得到DUM神经元细胞, 运用膜片钳技术记录DUM神经元细胞电压门控Na+电流。【结果】分离得到的DUM神经元细胞状态良好, 具有DUN神经元典型的梨状形态和表面特征。以膜片钳全细胞方式记录到的Na+电流符合钠通道电流特征。【结论】IA型胶原酶消化得到美洲大蠊DUM神经元细胞的方法可靠, 能稳定地记录到Na+电流。本文描述的方法为昆虫神经细胞的电生理机制研究提供一个可用的实验模型。     

Reconfiguration of a Multi-oscillator Network by Light in the Drosophila Circadian Clock

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电刺激兔脑干中缝背核引起呼吸易化效应的观察

本文在30只全麻、制动、断双侧迷走神经的家兔上,记录一侧膈神经放电,观察了电刺激脑干中缝背核(Nucleus Raphe Dorsalis,NRD)所诱发出的呼吸效应。1.施以6—10s 长串电脉冲刺激(波宽0.3ms,频率100Hz,波幅4—6V),诱发出了强的呼吸易化效应,使呼吸加深加快。2.吸气相给予0.4s 短串电脉冲刺激可以明显的延长吸气相,用0.15mA 强度刺激,落位在吸气相的2/3时效应最明显。3.呼气相短串电脉串刺激可规律地使呼气时程缩短,促进呼气向吸气的位相转换,诱发此效应出现的强度阈值在呼气相中逐渐降低。     

条背萤成虫与幼虫发光器的超微结构观察

对水生萤火虫——条背萤Luciola substriata(Gorham)成虫和幼虫发光器的超微结构进行研究。结果表明,成虫发光器由明显的2层组成:反射层和发光层。反射层由排列紧密的“尿酸囊泡”构成,具有发达的气管结构,对光起反射作用;发光层由大量发光细胞构成,内含典型的发光颗粒、线粒体、内质网及大量糖原,该层通过发光细胞胞质内的生化反应而发光。2层均由非细胞层膜包被,间距25~30μm。发光器腹节由外向内依次为表皮、发光层、反射层和内部细胞层。幼虫发光器球形,由背射层和发光层构成,由非细胞层膜包被。背射层由单层柱状细胞构成,内含大量“尿酸囊泡”。发光层细胞膜相互绞缠,含有2种类型的发光颗粒:“致密”型和“凋亡”型,含有大量的线粒体和无定形颗粒,发光细胞之间分布着大量的气管、微气管及神经末梢,可观察到神经突触。与条背萤相比,陆生种成虫反射层和发光层均无非细胞层膜包被,2层间无明显间距,发光颗粒形状不规则,气管通常形成2分支;陆栖种幼虫发光层形状差异较大,背射层由单层或2~4层细胞构成;相似点在于,成虫发光器都由均由反射层和发光层构成,发光细胞内都含发光颗粒、线粒体及大量糖原,都具有发达的气管结构,发光颗粒相似。幼虫发光器都由背射层和发光层构成,都具有发达的气管和直接的神经支配,发光颗粒相似,都由非细胞层膜包被。     

电刺激杏仁复合体诱发心律失常及其下行神经通路的初步探讨

电刺激杏仁复合体能诱发心律失常。心律失常的类型为心动过缓伴室性或结性期外收缩。刺激杏仁复合体不同亚核均能诱发心律失常,不同类型的心律失常在核内具有相应的代表点。心律失常发作与杏仁局部区域诱发的爆发性后放电有关。推测杏仁复合体内神经元过度激活可能通过杏仁-迷走神经运动背核及杏仁-下丘脑外侧区等通路下行,使心率减慢、房室传导阻滞而导致心律失常。     

大鼠背根节神经元后超极化中Ca~(2 )激活K~ 电流的蜂毒明肽敏感成分I_(AHP)

为了明确大鼠背根节（DRG）神经元中存在慢的Ca2 激活K 电流成分,本实验在新鲜分散的DRG神经元胞体上,采用全细胞电压箝技术,给予DRG神经元一定强度的去极化刺激,记录刺激结束后30ms时的尾电流幅度。结果发现：（1）随着去极化时间从1ms延长至180ms时,尾电流幅度由9．3±2．8pA逐渐增大至64.1±3．4pA（P＜0．001）;（2）当去极化结束后的复极化电位降低时,尾电流幅度先逐渐下降到零,然后改变方向,逆转电位约为-63mV;（3）细胞外施加500μmol／LCd2 或细胞内液中施加11mmol／LEGTA时尾电流明显减小甚至完全消失;（4）尾电流中慢成分的幅度在细胞外给与200nmol／L蜂毒明肽后,减小了约26.32±3。9％（P＜0。01）;（5）细胞外施加10mmol／LTEA,可明显降低尾电流中的快成分。结果提示,在DRG神经元启超极化中存在Ca2 激活K 电流的蜂毒明肽敏感成分──IAHP。