Organotypic Brain Slice Cultures for Functional Analysis of Alcohol-Related Disorders: Novel Versus Conventional Preparations

Assessment of long-term alterations in neural function and phenotype has usually involved culture techniques that utilize dissociated preparations. Recently, we have approached such topics in alcohol research by using brain slice cultures, also known as explant or organotypic preparations. In this symposium presentation, two preparations will be discussed, and examples of the particular advantages of these preparations will be presented in relation to alcohol research. First, we use the hippocampal explant preparation for assessment of long-term alterations in N -methyl-D-aspartate receptor (NMDAR) function due to chronic ethanol exposure and subsequent withdrawal. This preparation displays many synaptic, structural, and enzymatic phenotypes indicative of normal neural preparations. Patch clamp recordings reveal NMDAR-mediated excitatory postsynaptic current (EPSC) elicited upon stimulation of Schaffer collateral fibers and recorded from CA1 pyramidal cells. Long-term ethanol exposure followed by subsequent withdrawal resulted in a specific enhancement of NMDAR-mediated synaptic responses which preceded the expression of epileptiform events that occurred after prolonged withdrawal periods. Second, we describe a novel explant preparation, derived from horizontal slices of the entire forebrain and midbrain of the rat. These long-term explants displayed multiple normal phenotypes including Nissl, AChE, TH, and GFAP staining. Electrophysiologically, these explants displayed a functional corticostriatal pathway recorded with field and patch clamp techniques and elicited by synaptic stimulation. Taken together, these explant preparations display utility for long-term study of ethanol effects on neural systems, especially relating to withdrawal hyperexcitability as well as systems involved in drug-seeking behavior.     

雪旺氏细胞脑内移植前的体外培养

目的 介绍新生鼠Schwann细胞培养方法，为今后临床Schwann细胞移植提供实验基础。方法 采用坐骨神经组织块反复种植同时加刺激因子forskolin和BTI培养Schwann细胞。相差显微镜形态学连续观察及间接免疫荧光染色显示Schwann细胞胶质特异性标志蛋白S100表达和BrdU掺入。结果 形态学连续观察和S100间接免疫荧光染色都显示，Schwann细胞纯度可达98％-99％；BrdU掺入显示Schwann细胞增殖活性很强。结论 坐骨神经组织块反复种植同时加刺激因子forskolin和BTI不失为Schwann细胞培养好方法。此方法简便、快速，可在短时间内获得大量高纯度、有活性的Schwann细胞。     

改良的软骨组织块培养法

为软骨的组织工程的临床应用寻求一种与之相适应的体外扩增种子细胞的方法。将传统的组织块培养法进行改良 ,在置入培养皿前先以 0 .2 5 %胰蛋白酶、0 .0 8?TA及 0 .1%睾丸透明质酸酶消化软骨组织块 (实验组 ) ,以传统的组织块培养法为对照组进行培养。结果 :在培养的第 3天 ,实验组贴壁率为 88.89% (32 / 36 ) ,对照组为 6 8.89% (2 3/ 36 ) (P <0 .0 5 ) ;培养后的第 30天 ,实验组的细胞簇形成阳性率为 10 0 % (12 / 12 ) ,而对照组为 0 (0 /12 ) (P <0 .0 1)。结论 :改良的软骨组织块培养法优于传统的组织块培养法 ,更适合于软骨的组织工程临床应用     

Evidence for the existence of galanin receptors on cultured astrocytes of rat CNS: colocalization with cholinergic receptors

The cellular localization of binding sites for [¹²⁵I]galanin was studied in explant cultures of rat neocortex, cerebellum, locus coeruleus and spinal cord by means of autoradiography. Binding sites for the peptide were observed on a great number of astrocytes in all CNS regions studied. In addition to astrocytes, many neurones were intensely labelled by [¹²⁵I]galanin. Binding of [¹²⁵I]galanin (10⁻⁸ M) to both astrocytes and neurones was markedly reduced or inhibited by the unlabelled peptide at high concentration (10⁻⁶ M), suggesting `specific' binding of the radioligand. Evidence for the colocalization of galanin and cholinergic receptors on astrocytes was provided by combined autoradiographic and immunohistochemical studies. Many astrocytes were labelled by [¹²⁵I]galanin and immunostained with antibodies to either muscarinic or nicotinic receptors. Electrophysiological studies revealed that addition of galanin (10⁻⁹ to 10⁻⁷ M) to the bathing fluid caused a dose-dependent hyperpolarization of the majority of astrocytes studied. When galanin (10⁻⁸ M) and the cholinergic agonists muscarine and nicotine (10⁻⁶ M) were tested on the same astrocyte, all three compounds induced a hyperpolarization, suggesting a colocalization of functional galanin and cholinergic receptors on the glial membrane.     

Pituitary adenylate cyclase-activating peptide (PACAP) and PACAP type 1 receptor expression in regenerating adult mouse and rat superior cervical ganglia in vitro

Pituitary adenylate cyclase-activating polypeptide (PACAP), a regulatory peptide belonging to the vasoactive intestinal peptide (VIP) family, is widely distributed in the central and peripheral nervous system. Recent studies have shown that PACAP expression is upregulated in sensory neurons in response to axonal injury. Here we report that PACAP and PACAP type 1 receptors are located in rat and mouse superior cervical ganglia (SCG). PACAP-immunoreactivity (-IR) was demonstrated in preganglionic fibers, whereas only occasional PACAP-IR cell bodies could be observed. In situ hybridization histochemistry using ³⁵S-labeled deoxyribonucleotide probes confirmed that PACAP mRNA was present only in occasional cell bodies. In contrast, PACAP type 1 receptor mRNA was expressed in virtually all cell bodies within the ganglia. After removal and culturing of the SCG for 24 h, there was a marked increase in PACAP mRNA, whilst PACAP type 1 receptor mRNA expression appeared to be downregulated in most nerve cell bodies except for a few scattered neurons displaying a strong upregulation. The total specific binding of PACAP to isolated SCG membranes as assayed by [¹²⁵I]PACAP-27 binding showed an increase in SCG cultured for 48 h. PACAP-27 neither affected axonal outgrowth from the cultured SCG nor the survival of cells within the SCG. We conclude that PACAP and PACAP receptors are rapidly upregulated in sympathetic ganglia in response to axonal injury and that PACAP may play a role during nerve regeneration.     

Nucleosides influence the myelination of dordal root ganglion cells in vitro; a quantitative comparison

α-Modified minimal essential medium (αMEM) is the most useful commercial medium for survival and myelin formation of dorsal root ganglia (DRGs). When components of αMEM are compared with those of other commercial media, nucleosides are found only in αMEM. We examined the effects of nucleosides on myelin formation in rat DRG cultures. When myelin formation was viewed quantitatively, cultures using αMEM withy nucleosides yielded 2- to 3-fold more myelin segments than cultures using αMEM without nucleosides.     

Electrophysiological characteristics of neurons in neocortical expiant cultures

Summary We examined the electrophysiological and morphological properties of neocortical neurons maintained in expiant cultures prepared from the parietal cortex of newborn Sprague-Dawley rats. After 3–6 weeks in vitro, cultures showed regional differences in cellular density reminiscent of cortical layering, and an abundance of axonal processes. Pyramidal-shaped neurons with spinous dendrites were the dominant elements revealed by Lucifer yellow injections. Intracellular recordings revealed that many electrophysiological properties of neurons in the explants resembled those of neocortical neurons in vivo and in slice preparations. In response to depolarizing current injection, neurons in the expiants showed the same three patterns of repetitive firing described in neocortical slices, as well as a similar array of responses. Spontaneous synaptic potentials were recorded from all neurons and complex PSPs were evoked in response to focal extracellular stimulation. GABAa receptors mediated a significant component of the evoked responses. Fifteen of sixty neurons generated action potentials that arose spontaneously from resting potentials. Neurons in many slices generated large, prolonged depolarizing potentials that reflected coordinated synaptic activity within the expiants. These results underscore the usefulness of the neocortical explant as a valuable model for studying aspects of the behavior of circuits of cortical neurons.     

孔源性视网膜脱离裂孔位置与手术疗效的关系

目的:观察研究巩膜外加压术手术治疗上下方裂孔的孔源性视网膜脱离的疗效差异。方法:回顾性分析接受巩膜外加压术治疗的140例140眼孔源性视网膜脱离患者中裂孔位于上方者74眼、裂孔位于下方者66眼的术后疗效差异。结果:行巩膜外加压术治疗的下方裂孔的孔源性视网膜脱离患者的视网膜复位率要低于上方裂孔的孔源性视网膜脱离者。结论:视网膜裂孔位于上方的患者行巩膜外加压术治疗的疗效要好于裂孔位于下方者。     

原代人牙周膜成纤维细胞的培养及培养方法的改进

目的：探讨如何提高人牙周膜成纤维细胞原代培养的成功率。方法：组织来源于11～15岁青少年因正畸而拔除的前磨牙，刮取牙根中1／3的牙周膜组织，组织贴壁培养法进行培养采用冠根分离控制取材中的污染，首次传代前进行细胞分散的方法来提高传代的成功率，并对培养细胞的形态及超微结构进行了观察。结果：经免疫组化染色证实所培养的细胞为牙周膜成纤维细胞，通过冠根分离法可有效的控制组织污染，首次传代前进行细胞分散可提高细胞传代成功率，从而使细胞培养成功率提高。结论：获得人牙周膜成纤维细胞，培养成功率在15％～20％。