电针可增强大鼠脑内前脑啡肽原mRNA的表达：原位杂交组织化学研究

本文采用原位杂交组织化学技术，结合计算机图象处理系统，对电针后大鼠脑内前脑啡肽原（ＰＰＥ）ｍＲＮＡ的表达进行半定量的观察。ＰＰＥｍＲＮＡ探针以半抗原的地高辛标记。电针１０小时之后，ＰＰＥｍＲＮＡ阳性信号的强度和神经元内合ＰＰＥｍＲＮＡ的面积在许多与痛和镇痛有关的核团明显增加，如尾核，伏核，视前区，中脑的脚间核，导水管周围灰质，黑质，红核等。表明电针促进了ＰＰＥｍＲＮＡ在大鼠脑内的表达。这可能是针刺具有累积和长期效应的机制之一。     

疼痛及针刺镇痛时孤啡呔受体mRNA的变化

目的：观察大鼠在福尔马林致痛及针刺镇痛时孤啡肽受体ｍＲＮＡ在一些与镇痛有关核团的变化情况。方法：采用原位杂交组织化学技术。结果：电针后１０ｈ，导水管周围腹侧区、中缝背核及中缝大核内孤啡肽受体ｍＲＮＡ阳性神经元数增多；而在大鼠脚掌注射福尔马林后，上述核团内孤啡肽受体ｍＲＮＡ阳性神经元数却明显减少；电针并注射福尔马林，脑内孤啡肽受体ｍＲＮＡ水平介于单用电针和福尔马林之间。结论：电针能促进孤啡肽受体的合成而伤害性刺激抑制孤啡肽受体的合成。     

电针后大鼠中枢多巴胺D1和D5受体mRNA的变化

应用原位杂交，放射自显影结合计算机图像处理技术检测电针（ＥＡ）后１０ｈ及２４ｈ时大鼠中枢多巴胺（ＤＡ）Ｄ１和Ｄ５受体信使ＲＮＡ（ｍＲＮＡ）的变化。结果显示，ＥＡ后１０ｈ脊髓Ｄ１和Ｄ５受体ｍＲＮＡ有明产高，２４ｈ时进一上不增高；而脑内Ｄ１和Ｄ５受体ｍＲＮＡ需至２４ｈ时才有显著增高，结果提示，在转录水平，ＥＡ可加强大鼠中枢Ｄ１和Ｄ５受体基因的表达，使这些受体合成增加。     

CCK—8及损毁MFB对腹侧被盖区和伏核多巴胺,CCK—8含量的影响

应用荧光分光光度法和放射免疫法，在以６－羟基多巴胺（６－ＯＨＤＡ）单侧损毁内侧前脑束（ＭＦＢ）制备的偏侧帕金森病（ＰＤ）大鼠模型身上，测定了腹侧被盖区（ＶＴＡ）和伏核（Ａｃｂ）中多巴胺（ＤＡ）和八胺胆囊收缩素（ＣＣＫ－８）的含量，并测定了ＴＶＡ和Ａｃｂ区微量注射ＣＣＫ－８对正常大鼠ＤＡ含量的影响。结果如下：ＰＤ大鼠模型损毁侧ＶＴＡ和Ａｃｂ的ＤＡ和ＣＣＫ－８的含量与健康及对照组相比均减少（Ｐ〈０．０     

鼠尾伤害性刺激与颧Liao穴电针后弓状核—导水管周围灰质—延髓头端腹？…

成年ＳＤ大鼠分成对照，尾部电流所致伤害性刺激，面部颧Ｌｉａｏ穴电针三组，分别观察弓状核，中脑中央灰质和延髓头端腹内侧区原位杂交后在神经元中前脑啡肽原（ＰＰＥ）ｍＲＮＡ的表达与Ｆｏｓ免疫阳性神经元的分布，痛刺激后上述各部位的ＰＰＥｍＲＮＡ在神经元内均有不同程度表达，但当颧Ｌｉａｏ穴电针时表达更明显，细胞数也较多。尤其在延髓头端腹内侧区更显著，而在对照动物仅有面神经核等与运动有关核闭出现表面，在ＰＰＥ     

免疫应答期间大鼠部分脑区内乙酰胆碱酯酶活性的变化

用绵羊红细胞（ＳＲＢＣ）免疫大鼠，在免疫后第３天至第６天，分别检测大鼠下丘脑、丘脑、中脑和脑桥内乙酰胆碱酯酶（ＡＣｈＥ）活性的变化。其中丘脑、中脑和脑桥在免疫后第３至６天，ＡＣｈＥ活性与对照组比较均无显著差异。而下丘脑内ＣＡｈＥ活性从第３天开始下降，至第６天均明显降低．本实验说明，在免疫应答期间，下丘脑中ＡＣｈＥ活性降低，由此提示体液免疫应答可影响下丘脑中乙酰胆碱（ＡＣｈ）的含量。我们以往的实验表明，中枢ＡＣｈ可调控体液免疫功能。综合两者结果，可以推断中枢ＡＣｈ与免疫之间存在相互作用。     

AVP_(4-8)增加海马组织内NGF mRNA和蛋白含量

本文采用了Ｎｏｒｔｈｅｒｎ杂交和ＥＬＩＳＡ法，观察了神经肽ＡＶＰ４－８对大鼠海马组织中神经生长因子（ＮＧＦ）ｍＲＮＡ和蛋白水平的影响，结果显示：给大鼠皮下注射ＡＶＰ４－８１２ｈ后，海马组织中ＮＧＦｍＲＮＡ的水平明显高于生理盐水对照组。而给大鼠皮下注射精氨酸加压素（ＡＶＰ）和催产素（ＯＴ）对ＮＧＦｍＲＮＡ的水平均无显著影响。另外，用ＡＶＰ４－８孵育离体大鼠海马组织切片，能使ＮＧＦ蛋白表达量升高，且在４．５ｈ左右达到高峰。     

神经营养素-3及其受体在小鼠脾的分布和基因表达

目的　在蛋白和基因水平进行神经营养素－３ （ＮＴ－３） 及其高亲和力受体ＴｒｋＣ的小鼠脾定位研究。方法　免疫组织化学和原位杂交。结果　脾内存在ＮＴ－３、ＴｒｋＣ免疫反应阳性（－ｉｒ） 细胞和ｍＲＮＡ。ＮＴ－３－ｉｒ细胞属于网状细胞, 主要散布于动脉周围淋巴鞘（ＰＡＬＳ）、淋巴小结和血管内壁。ＴｒｋＣ－ｉｒ细胞则分布于边缘区和红髓, 细胞性质有待确定。原位杂交实验证实, ＮＴ－３ｍ ＲＮＡ 和ＴｒｋＣｍＲＮＡ 分别与相应的免疫反应阳性细胞有一致的分布。免疫组化和原位杂交对照实验均为阴性。结论　ＮＴ－３ 和ＴｒｋＣ能够被脾内不同的细胞合成, 提示ＮＴ－３ 有可能具有调节脾功能的作用。     

癫痫状态大鼠海马GAD67mRNA,GABA—TmRNA表达水平的动态观察

为了研究癫痫状态下大鼠海马区ＧＡＤ６７ｍＲＮＡ、ＧＡＢＡ－ＴｍＲＮＡ的表达水平，采用马桑内酯（ｃｏｒｉａｒｉａｌａｃｔｏｎｅ，ＣＬ）侧脑室注射所致大鼠癫痫状态模型，动态观察ＣＬ致痫前后大鼠海马ＧＡＤ６７ｍＲＮＡ、ＧＡＢＡ－ＴｍＲＮＡ表达水平的变化，同时观察了动物行为学改变及脑电图变化。实验结果显示，侧脑室注射ＣＬ后约１５分钟，大鼠海马及皮质ＥＥＧ出现阵发性棘慢波及高波幅慢波；ＣＬ注射后１５～３０分钟，实验鼠均出现明显的连续发作性四肢抽动、尖叫等症状，且持续６～８小时。ＣＬ侧脑室注射后３０分钟、１小时、２小时、４小时或８小时各时限点组大鼠海马ＧＡＤ６７ｍＲＮＡ表达水平均低于正常对照组。ＣＬ注射后３０分钟组大鼠海马ＧＡＢＡ－ＴｍＲＮＡ表达水平高于正常对照组，但３０分钟后各时限组ＧＡＢＡ－ＴｍＲＮＡ表达水平与正常对照组比较增加不明显。提示ＣＬ所致癫痫持续状态的后期，大鼠海马ＧＡＤ６７ｍＲＮＡ、ＧＡＢＡ－ＴｍＲＮＡ表达信号的减弱，可能与致痫因素及癫痫持续后期合并的缺血、缺氧性脑损害致使海马区ＧＡＤ、ＧＨＢＡ－Ｔ免疫反应阳性细胞死亡有关。     

不同氨基酸受体拮抗剂对缺血纹体CCK—8释放的影响

目的：研究不同兴奋性氨基酸受体拮抗剂（ＭＫ８０１，Ｓｐｅｒｍｉｎｅ，ＤＮＱＸ）对大鼠脑缺血时纹体缩胆囊肽－８（ＣＣＫ－８）释放的影响。方法：用微透析和ＲＩＡ法测定半球缺血时纹体ＣＣＫ－８的释放。结果：脑缺血时纹体ＣＣＫ－８释放明显增高，缺血前分别给予三种拮抗剂，都明显地降低缺血时纹体ＣＣＫ－８的不正常释放。结论：表明脑缺血时的ＣＣＫ－８和兴奋性氨基酸（ＥＡＡ）之间有协同的相互作用，抑制ＣＣＫ－８释放增高，可能是兴奋性氨基酸受体拮抗剂保护缺血大脑的机制之一。     

Central D-Ala2-Met5-enkephalinamide mu/delta-opioid receptor activation blocks behavioral sensitization to cholecystokinin in CD-1 mice

The present investigation revealed that intraventricular administration of the anxiogenic substance CCK-8S (50 ng) decreased responding for previously rewarding brain stimulation (intracranial self-stimulation; ICSS) and subsequently increased brain stimulation threshold determinations from the dorsal aspects of the ventral tegmental area (VTA) immediately following CCK administration. While central administration of the mixed mu/delta opioid receptor agonist D-Ala(2)-Met(5)-enkephalinamide (DALA; 1 microg) was ineffective in abrogating CCK induced ICSS deficits during the immediate post-stressor interval, DALA restored ICSS brain stimulation thresholds to basal values 24, 48 and 168 h following CCK challenge. At 18 days following the initial 50 ng CCK-8S and/or DALA challenges, mice were exposed to a previously determined non-anxiogenic dose of CCK-8S (5 ng). Among mice which received an intervening dose of saline following the 50 ng CCK-8S challenge, depressed ICSS responding and elevated brain stimulation thresholds were evident during the immediate (Day 18), 24- (Day 19) and 48-h (Day 20) test sessions relative to mice that received an intervening dose of DALA on Day 1. These data imply that while CCK induces relatively protracted and exaggerated behavioral disturbances, mu/delta opioid-receptor activation may block CCK-induced behavioral sensitization and change the course of psychopathology.     

鼠尾伤害性刺激与颧髎穴电针后弓状核-导水管周围灰质-延髓头端腹内侧区的前脑啡肽原mRNA的表达及与Fos蛋白共存

成年SD大鼠分成对照、尾部电流所致伤害性刺激、面部颧髎穴电针三组，分别观察弓状核、中脑中央灰质和延髓头端腹内侧区原位杂交后在神经元中首脑啡肽联原(PPE)mRNA的表达与Fos免疫阳性神经元的分布。痛刺激后上述各部位的PPEmRNA在神经元内均有不同程度表达，但当颧髎穴电针时表达更明显，细胞数也较多。尤其在延髓头端腹内侧区更显著，而在对照动物仅在面神经核等与运动有关核团出现表达，在PPEmRNA与Fos免疫组化相结合实验中．痛组与针刺组动物中皆可观察到一定比例数神经元的共存，及单独Fos和单独EnkmRNA阳性神经元的存在。     

CCK(B) receptor antagonist L365,260 potentiates the efficacy to and reverses chronic tolerance to electroacupuncture-induced analgesia in mice

Cholecystokinin octapeptide (CCK-8) is a physiological antagonist of endogenous opioids in the central nervous system (CNS). Our previous work has shown that CCK-8 plays an important role in the development of tolerance to morphine analgesia and electroacupuncture (EA) analgesia in the rat. The present studies were designed to examine whether the CCK(B) receptor is involved in the modulation of EA analgesia and the development of EA tolerance in mice. The latency to flick the tail in the radiant heat was used as index to assess the efficacy of EA analgesia. Subcutaneous (s.c.) injection of the CCK(B) receptor antagonist L365,260 produced a dose-dependent (0.125-2.0 mg/kg) potentiation of the analgesia induced by 100 Hz EA, with a maximal effect occurred at 0.5 mg/kg. In addition, L365,260 (0.5 mg/kg) significantly reversed chronic tolerance to 100 Hz EA in mice. These results suggest that the CCK(B) receptor might play a role in the tonic inhibition of 100 Hz EA-induced analgesia and in the mediation of chronic tolerance to 100 Hz EA in mice. The results opened a way for further investigation of the function of CCK-8 in pain modulation using inbred strains of mice.     

Endogenous opioids modulate the effect of cholecystokinin on insulin release in dogs

Recently we have demonstrated in dogs and man that endogenous opioids participate in the regulation of pancreatic endocrine function following the ingestion of a meal. Since intestinal hormones such as cholecystokinin (CCK) are also released by the presence of nutrients in the gastrointestinal tract and participate in the postprandial stimulation of pancreatic endocrine function, an interaction between CCK and endogenous opioids seems possible. The present study was designed to examine this further. In a group of 8 conscious dogs the octapeptide of CCK was infused intravenously in its sulfated (CCK-8S) or nonsulfated (CCK-8NS) form and in addition the tetrapeptide of CCK (CCK-4) was given at increasing infusion rates of 50, 200 and 500 pmol/kg . h, respectively. The experiments were performed during a background infusion of saline to assess the effect on basal insulin and during a background infusion of glucose (0.2 g/min) to determine the effects on stimulated insulin release. The effect of endogenous opioids was examined by addition of the opiate-receptor antagonist naloxone. The studies demonstrate that in the basal state CCK-8S has no stimulatory effect on insulin secretion unless naloxone is added indicating that endogenous opioids help to prevent insulin secretion in the absence of elevated glucose levels. During i.v. glucose naloxone reduced the stimulatory effect of CCK-8S at 50 and 200 pmol/kg . h and that of CCK-4 at 50 pmol/kg . h. Infusion of CCK-8S and CCK-4 at 500 pmol/kg . h had no effect on glucose-stimulated insulin levels, however, the addition of naloxone elicited a significant stimulatory effect. These data demonstrate stimulatory as well as inhibitory effects of endogenous opioids depending on the dose of CCK-8 and -4. CCK-8NS reduced glucose-stimulated insulin release already at the lowest dose of 50 pmol/kg . h. This was reversed to a stimulatory effect with the addition of naloxone. These data demonstrate that the interaction between CCK-8 and -4 and endogenous opioids on prestimulated insulin secretion is much more dependent on the dose of CCK - low doses induce stimulatory and high doses inhibitory mechanisms via endogenous opioids. In view of previous in vitro and in vivo studies with exogenously infused opiate-active compounds it might be speculated that increasing doses of CCK elicit a parellel increase in the release of endogenous opioids which might be responsible for some but certainly not all of the effects observed recently for the action of naloxone in the post-prandial state.     

癫痫易感大鼠脑内CCK—8肽含量,CCK基因结构及CCK—mRNA表达初探

对听源性癫痫易感大鼠P_(77)PMC及对照Wistar大鼠发育中脑内基础CCK-8肽含量、mRNA表达水平及胆囊收缩素(cholecystokinin,CCK)基因结构进行初探。提示:(1)P_(77)PMC在发育中,成年脑内基础CCK-8肽显著高于对照;(2)Northern印迹杂交示P_(77)PMC组脑CCK-mRNA与对照无显著差异;(3)CCK DNA结构在两组大鼠未发现RFLPs不同。证实两系大鼠脑内CCK系统存在差异,其与癫痫易感性的关系尚待进一步研究。     

Receptor selectivity of cholecystokinin effects on mesoaccumbens dopamine neurons

Extracellular recording techniques were combined with antidromic stimulation to examine the effects of C-terminal cholecystokinin (CCK) fragments and CCK antagonists on the activity of identified mesoaccumbens dopamine (MADA) neurons in chloral hydrate-anesthetized rats. These experiments were designed to determine the receptor selectivity of sulfated CCK octapeptide (CCK-8S) effects on MADA cells. Neither CCK tetrapeptide (CCK-4) nor unsulfated CCK octapeptide (CCK-8U) significantly altered MADA cell basal firing rate or responsiveness to the inhibitory effects of the D2 DA agonist quinpirole. As reported previously for ventral tegmental area DA cells, CCK-8S produced increases or decreases in the firing rate of most MADA cells sampled. CCK-8S also enhanced the sensitivity of MADA neurons to quinpirole-induced inhibition. This increase in sensitivity to quinpirole was blocked by pretreatment with the nonselective CCK receptor antagonist proglumide and the preferential CCK-A receptor antagonist CR 1409 but not by the preferential CCK-B receptor antagonist L-365,260. The inactivity of CCK-4 and CCK-8U in these tests and the results with the antagonists suggest that the effects of CCK-8S on MADA neuronal activity are mediated by CCK-A receptors.     

Brain CCK-B receptors mediate the suppression of dopamine release by cholecystokinin

The sulfated octapeptide of cholecystokinin (CCK-8S) and CCK fragments were administered to mice to determine the subtype and central versus peripheral location of the CCK receptor that modulates dopamine release in the neostriatum. Dopamine release was decreased when unsulfated CCK (CCK-8U) or the butoxycarbonyl tetrapeptide of CCK (t-boc-CCK-4) was infused into the brain ventricles but not when injected subcutaneously. These CCK fragments bind to the brain-type (CCK-B) but not alimentary-type (CCK-A) receptor. Centrally or peripherally administered CCK-8S also lowered dopamine release and this action was not blocked by the selective CCK-A receptor antagonist, L 364,718. The increase in dopamine release following amphetamine administration was attenuated by central injections of t-boc-CCK-4, CCK-8U, or CCK-8S, and this action of CCK-8S was not prevented by L 364,718. These data are the first to demonstrate that CCK-B receptors in brain mediate the suppression of dopamine release by cholecystokinin, especially when release is augmented. CCK-B receptor agonists should be useful for the treatment of psychiatric conditions that result from hyperactive dopamine neurons.     

Measurement and characterization of neuronal cholecystokinin using a novel radioreceptor assay

This study describes a novel radioreceptor assay (RRA) for cholecystokinin (CCK) which is the first to measure and characterize brain CCK using a technique not dependent on the generation of peptide antibodies. The CCK RRA utilizes the mouse cerebral cortex CCK receptor as the binding source and [125I]BH-CCK-8 as the radiolabelled probe. [125I]BH-CCK-8 bound to the central CCK receptor with a Kd of 1.82 nM and a Bmax of 1.21 pmol/g tissue. Unlabelled CCK-8 displaced the specific binding of [125I]BH-CCK-8 with an inhibition constant of 3.84 nM. CCK was extracted (90% methanol) from discrete brain regions (mouse) and quantified using the CCK RRA. The amygdala contained the highest concentration of CCK (394 +/- 21 pmol/g tissue), followed by the olfactory bulbs (306 +/- 19 pmol/g tissue) and cerebral cortex (298 +/- 21 pmol/g tissue). Moderate levels of CCK were found in the hippocampus (212 +/- 18 pmol/g tissue), striatum (146 +/- 15 pmol/g tissue) and hypothalamus (129 +/- 9 pmol/g tissue). Low levels of CCK were recorded in the pons (45 +/- 5 pmol/g tissue), medulla (41 +/- 3 pmol/g tissue) and spinal cord (29 +/- 3 pmol/g tissue), whilst no CCK was detected in the cerebellum. The molecular forms of CCK in amygdala, cerebral cortex and hypothalamus were characterized using RRA in conjunction with HPLC. CCK-8 was identified as the major molecular form (88%, 94% and 91% of total CCK activity in amygdala, cortex and hypothalamus, respectively) with a smaller component attributable to CCK-4 (8%, 5% and 6% of the total CCK activity).     

Cholecystokinin‐8 antagonizes electroacupuncture analgesia through its B receptor in the caudate nucleus

Objectives: The analgesic effect of electroacupuncture (EA) stimulation has been proved. However, its mechanism of action is not clear. It has been well‐known that cholecystokinin‐8 (CCK‐8) is a neuropeptide which is mainly related to the mediation of pain. The caudate nucleus was selected to determine if the release of CCK and the neural activity in this nucleus were involved in producing EA analgesia. Materials and Methods: Radiant heat focused on the rat‐tail was used as the noxious stimulus. The pain threshold of rats was measured by tail‐flick latency (TFL). EA stimulation at the bilateral Zusanli (ST 36) acupoints of rats was used to investigate the effects of EA analgesia. The electrical activities of pain‐excited neurons (PEN) and pain‐inhibited neurons (PIN) in the caudate nucleus were recorded with a glass microelectrode. The present study examined the antagonistic effects of the intracerebral ventricular injection of CCK‐8 on EA analgesia and reversing effects of CCK‐B receptor antagonist (L‐365,260) injection into the caudate nucleus on CCK‐8. Results: The radiant heat focused on the tail of rats caused an increase in the evoked discharge of PEN and a reduction in the evoked discharge of PIN. EA stimulation at the bilateral ST 36 acupoints of rats resulted in the inhibition of PEN, the potentiation of PIN, and prolongation of TFL. The analgesic effect of EA was antagonized when CCK‐8 was injected into the intracerebral ventricle of rats. The antagonistic effect of CCK‐8 on EA analgesia was reversed by injection of CCK‐B receptor antagonist (L‐365,260) into the caudate nucleus of rats. Conclusions: Our results suggest that CCK‐8 antagonize EA analgesia through its B receptor.