胃动素对大鼠海马神经元放电活动的作用机制

目的：研究胃动素对大鼠海马胃扩张神经元放电活动影响的机制。方法：采用微电极细胞外记录方法在体观察胃动素对胃扩张（GD）神经元放电活动作用的机制。结果：①海马CA1区内的39个胃扩张兴奋性（GD—E）神经元中有21个（54％）对胃动索呈现兴奋性效应;在16个胃扩张抑制性GD—I）神经元中有10个神经元（63％）表现为兴奋效应,4个神经元放电频率降低;②海马CA1区注射胃动素后,33个GD—E神经元中有22个（67％）表现为兴奋性效应。注射一氧化氮合酶（NOS）抑制剂左旋精氨酸甲酯（L-NAME）后再注射胃动素,神经元的放电频率由（3．98±0．54）Hz减小到（2．09士0．42）Hz（P〈0．05）,22个原对胃动素有反应的神经元减少到15个,对胃动素的兴奋性由67％下降到46％;同样,在GD-I神经元中,细胞放电频率由（2．57±0．48）Hz下降到（1．85±0．30）Hz（P〈O．05）。但NO前体左旋精氨酸（L-AA）可明显加强胃动素对海马神经元的放电活动,放电频率由3．25±0．49Hz增加到4．59±0．61Hz（P〈o．05）。胃动素兴奋性神经元也从26个（57％）增加到32个（70％）。同样,注射L-AA后胃动素使GD-I神经元放电频率由（3．51±0．41）Hz增加到（4．44±0．49）Hz（P〈0．05）;但L-AA的同分异构体6-右旋精氨酸（D—AA）对胃动素诱导海马神经元放电活动元改变（P〉0．05）。结论：L-AA可增强胃动素对海马胃扩张神经元的兴奋作用;而L—NAME减弱此作用。     

大电导钾通道对小鼠皮层神经元胞内游离钙和动作电位的影响

目的 探讨大电导钾通道（BK）对小鼠大脑皮层神经元胞内游离钙 ( [Ca2+]i） 和兴奋性的调节作用。方法 体外培养小鼠皮层神经元,用膜片钳技术观察BK特异性阻断剂IBERIOTOXIN对神经元 [Ca2+]i和动作电位频率的影响,用显微荧光测钙法观察IBERIOTOXIN对高钾条件下[Ca2+]i的影响。结果 生理状态下,IBERIOTOXIN灌流(100nmmol/L)对神经元细胞自发动作电位的频率、[Ca2+]i无显著影响;持续电刺激去极化后,IBERIOTOXIN灌流使动作电位频率增加,[Ca2+]i显著升高。高钾溶液(20mmol/L)引起神经元[Ca2+]i升高, 而IBERIOTOXIN灌流则使[Ca2+]i进一步显著升高。结论 BK对小鼠受持续去极化刺激或高钾条件时的神经元[Ca2+]i和兴奋性具有明显调节作用。     

大鼠三叉神经中脑核内PPD、PPE样阳性终末与PAG和PV共存神经元的联系

目的：观察大鼠三叉神经中脑核（Vme)神经元内磷酸激活的谷氨酰胺酶（PAG）和小白蛋白（PV）的共存关系，以及前强啡肽原（PPD）样和前脑啡肽原（PPE）样阳性科末与PAG和PV共存神经元之间的联系。方法和PV样免疫反应神经元，吻尾方向上几乎在其全长出现，多为大的假单极神经元，许多PAG样阳性神经元同时呈PV燕免疫反应，双标细胞占全部标记细胞的95％以上，在激光共聚焦显微镜下观察到，少量PPD或PPE样阳性终末围绕在PAG样和PV样双重标记的Vme神经元胞体周围，并与之形成密切接触，结论：在面口部本体感觉住处由Vme向更高一级神经元的过程中，PV可能和谷氨酸一起发挥着重要的作用，与此同时Vme神经元还可能接受中枢内强啡肽样和脑啡肽样神经终末对它的调控。     

BKCa通道对小鼠杏仁核锥体神经元兴奋性的影响

目的:观察大电导钙依赖性钾通道(large conductance calcium-activated potassium channels,BKCa)对小鼠外侧杏仁核锥体细胞神经元兴奋性的影响。方法:采用全细胞脑片膜片钳技术,记录小鼠杏仁核锥体神经元动作电位频率和幅度的变化。结果:在电流钳全细胞记录模式下输入一定强度的正电流诱导神经元发放动作电位,观察到灌流BKCa通道阻断剂iberiotoxin(IBTX100nmol/L)可显著增加动作电位发放频率,并缩短首个动作电位出现潜伏期;相反,BKCa通道激动剂NS1619(10μmol/L)可显著降低动作电位发放频率,并延长首个动作电位出现潜伏期。此外,BKCa通道参与单个动作电位后超极化电位(after hyperpolarizing potential,AHP)的形成。在电极内液中加入快速型钙离子螯合剂BAPTA(10mmol/L)可取消IBTX和NS1619对动作电位的影响。结论:BKCa通道对杏仁核锥体神经元的兴奋性具有重要的调节作用。     

高乌甲素在大鼠中脑导水管周围灰质的镇痛机制

目的观察高乌甲素(LA)能否通过上调中脑导水管周围灰质(PAG)中P2X3受体表达和功能,对烧伤疼痛产生镇痛作用。方法建立Ⅱ°烧伤大鼠模型,给予腹腔注射LA 6mg/kg·24h,Von Frey测痛仪检测机械痛阈值(MWT);Western blot检测P2X3受体蛋白表达;观察干预P2X3受体蛋白的表达和功能后对LA镇痛作用的影响。结果大鼠烧伤后MWT下降(P0.001),经LA治疗后MWT上升(P0.001),PAG中P2X3受体蛋白表达上调(P0.001)。下调P2X3受体蛋白表达后减弱LA镇痛作用;P2X3受体激动剂和拮抗剂分别增强和减弱LA的镇痛作用。结论 LA通过上调PAG中P2X3受体表达和增强P2X3受体功能,促进机体内源性镇痛,对烧伤疼痛大鼠产生镇痛作用。     

COX-2抑制剂对幼鼠海马CA1区锥体神经元钠通道的影响

目的：研究环氧合酶-2（cyclooxygenase-2,COX-2）抑制剂硝基苯一甲磺酸（NS398）对大鼠海马CAl区锥体神经元电压依赖性钠通道的影响,以及在幼鼠痫性发作中的作用。方法：出生后14d龄SD大鼠制作海马组织脑切片,脑片灌流液中灌流不同浓度NS-398,全细胞记录在阶跃模式（episod—ic）中,通过相应的刺激方案（protocol）,记录对电压依赖性钠通道电流密度及幅度的影响,观察对钠通道激活及失活曲线的影响。结果：①加入20μMol／L（μM）NS-398能明显抑制电流密度,而且在最大激活电位时抑制最明显（P〈O．01）,抑制呈电压依赖性,但是不能改变其最大激活电位;②加入20μmol／LNS-398不能明显改变钠通道的电压依赖性激活状态（P〉0．05）;③加入20Fmol／LNS-398,电压依赖性钠失活电流的失活曲线明显向负极化方向移动（左移5．2mV,P〈0．05）;④相同指令电压的刺激下,加入NS-398组的1／Lmax比正常组减小,NS-398能明显延长电压依赖性钠电流的失活时间。结论：COX-2抑制剂能抑制大鼠海马脑片CAl区锥体神经元电压依赖性钠通道,延长电压依赖性钠电流的失活时间;减少Na＋电流,延缓动作电位的发放和传播,降低神经元的兴奋性。     

COX-2抑制剂对幼鼠痫性发作海马神经元突触活动的影响

目的：观察环氧合酶-2（cyclooxygenase-2,COX-2）抑制剂硝基苯-甲磺酸（NS-398）对幼鼠痫样放电的作用及其对海马CA1锥体神经元突触电活动的影响,研究NS-398在幼鼠痫性发作中的作用。方法：用生后第14天龄SD大鼠制作海马组织脑切片,记录其CA1区锥体神经元场电位,以群峰电位（PS）个数和波幅作为指标来评价脑片放电的变化。给脑片用不同浓度青霉素,建立离体海马脑片痂样放电模型,在脑片灌流液中用不同浓度NS-398,观察对PS个数和波幅的影响。全细胞记录模式下,观察NS-398对海马CA1锥体神经元递质释放和突触活动的影响。分别记录自发性兴奋性突触后电流（sEPSC）和自发性抑制性突触后电流（sIPSC）,观察NS-398对其波幅和频率的影响。结果：NS-398浓度为10μmol／L时,对青霉素诱发的痼样放电没有多大的抑制效应;当浓度为20gmol／L时,有明显的抑制作用;为30μmol／L时抑制作用很强,明显降低PS的波幅和减少其频率。NS-398能明显抑制致痴大鼠海马锥体神经元sEPSC的频率,但是对其波幅及衰减时间没有明显的影响;同时NS-398能明显增强致痫大鼠海马脑片锥体神经元sIPSC的频率,明显延长sIPSC的衰减时间,对波幅影响不大。结论：COX-2抑制剂NS-398能减少sEPSC的放电和增强sIPSC的抑制功能,导致兴奋性神经递质的释放减少,降低神经元的兴奋性,从而抑制神经元异常放电。     

依托咪酯降低大鼠丘脑皮层脑片神经元活性

目的:先前的研究已经证明丘脑-皮层网络在全身麻醉药诱导意识消失过程中起着至关重要的作用。依托咪酯广泛用于临床麻醉,可以可逆性地引起意识消失(loss of consciousness,LOC)。我们的前期研究表明依托咪酯增强丘脑皮层网络γ-氨基丁酸(GABA)和谷氨酸能神经传递。然而,依托咪酯对丘脑皮层网络神经元动作电位的影响尚不清楚。本研究拟借助膜片钳技术观察依托咪酯对丘脑皮层网络神经元动作电位的影响。方法:20只健康(10～20 d)雄性SD大鼠制备含丘脑腹后内侧核(VPM)和初级躯体感觉桶状皮层(S1BF)的丘脑皮层离体脑片。运用全细胞膜片钳,观察不同浓度依托咪酯(3. 0μmol/L,6. 0μmol/L和12. 0μmol/L)对大鼠离体脑片丘脑皮层网络动作电位超摄值(OS)、升高其阈值(TP)和90%复极化动作电位时程(APD90)的影响。结果:依托咪酯降低丘脑VPM和S1BF动作电位的OS值,升高其TP,延长APD90。依托咪酯还降低了这两个脑区神经元动作电位的发放频率。与S1BF比较,依托咪酯对丘脑VPM神经元的抑制程度更大。结论:依托咪酯抑制丘脑-皮层神经元动作电位的产生,降低其发放频率,可能是其导致意识消失的机制之一。     

一氧化氮合成酶在培养海马神经细胞上的分布及其激活对细胞兴奋性的影响

目的：观察一氧化氮合成酶（NOS）在培养海马神经细胞上的分布情况和酶激活时对细胞兴奋性的影响。方法：NOS的分布情况采用免疫荧光标记方法，细胞兴奋性的变化采用膜片钳全细胞的模式来记录膜电位的变化。结果：发现两种结构型NOS包括nNOS和eNOS均分布在神经元上。另外，eNOS还分布在胶质细胞上。当给予NOS的底物L-精氨酸时，海马神经元的膜电位出现去极化，并产生动作电位。结论：以上结果显示NOS广泛分布在海马神经细胞中，当其激活时对海马神经元有兴奋作用。     

红藻氨酸对大鼠海马锥体细胞钙电流的影响

目的：应用膜片钳技术,观察红藻氨酸（KA）对大鼠海马锥体细胞ca2＋电流的影响,以研究癫痫的发病机制。方法：采用酶加机械分离法制备出生10～12d的大鼠海马锥体神经元标本,用全细胞膜片钳技术测定其生理学特性及观察KA对ca2＋电流的影响。结果：分离出的海马锥体细胞形态正常,有较长突起;用膜片钳技术证实,其保存了主要的离子通道活性。KA20μmol／L和100μmol／L的浓度均可使海马锥体细胞ca2＋电流峰值增大（n=8,P〈0．01）。结论：①大鼠海马锥体神经细胞具有明显的突起,细胞膜表面光洁、晕光好,适用于膜片钳实验研究;②KA使ca2＋内流增加,引起“Ca”超载”导致细胞毒性等一系列反应;③KA通过激活α-氨基羟甲基恶唑丙酸（AMPA）受体,诱发快速的兴奋性突触后电位（EPSP）,参与兴奋性突触传递。AMPA受体的激活可能是癫痫的发病机制之一。     

Perspective on the role of P2X-purinoceptor activation in human vas deferens contractility

The contractile actions of α,β-methylene ATP (α,β-meATP) and ATP and the effects of K(+) channel blockers in longitudinal and circular muscles of human vas deferens were investigated with a view to clarifying the functional importance of P2X(1)-purinoceptor activation and K(+) channels in modulating contractility of the tissues. The results provide an experiment-based perspective for resolving differing reports on purinergic activation of the tissues and uncertain roles of large-conductance Ca(2+)-activated K(+) (BK(Ca)) and voltage-gated delayed rectifier K(+) (K(V)) channels. α,β-Methylene ATP (3-100 μm) evoked suramin-sensitive contractions of longitudinal muscle but rarely of circular muscle. ATP (0.1-3 mm) less reliably activated only longitudinal muscle contractions. These were enhanced by ARL 67156 (100 μm), but a different ectonucleotidase inhibitor, POM 1, was ineffective. Both muscle types were unresponsive to ADP-βS (100 μm), a P2Y-purinoceptor agonist. Longitudinal muscle contractions in response to α,β-meATP were enhanced by FPL 64176 (1 μm), an L-type Ca(2+) agonist, TEA (1 mm), a non-specific K(+) channel blocker, 4-aminopyridine (0.3 mm), a selective blocker of K(V) channels, and iberiotoxin (0.1 μm), a selective blocker of BK(Ca) channels. Quiescent circular muscles responded to α,β-meATP reliably in the presence of FPL 64176 or iberiotoxin. Apamin (0.1 μm), a selective blocker of small conductance Ca(2+)-activated K(+) (SK(Ca)) channels had no effect in both muscle types. Y-27632 (1-10 μm) reduced longitudinal muscle contractions in response to α,β-meATP, suggesting involvement of Rho-kinase-dependent contractile mechanisms. The results indicate that P2X(1)-purinoceptor stimulation elicits excitatory effects that: (a) lead to longitudinal muscle contraction and secondary activation of 4-aminopyridine-sensitive (K(V)) and iberiotoxin-sensitive (BK(Ca)) K(+) channels; and (b) are subcontractile in circular muscle due to ancillary activation of BK(Ca) channels. The novel finding of differential action by P2X(1)-purinoceptor agonists in the muscle types has functional implication in terms of the purinergic contribution to overall contractile function of human vas deferens. The modulatory effects of K(V) and BK(Ca) channels following P2X(1)-purinoceptor activation may be pivotal in providing the crucial physiological mechanism that ensures temporal co-ordination of longitudinal and circular muscle contractility.     

Adenosine triphosphate-evoked currents in cultured dorsal root ganglion neurons obtained from rat embryos: desensitization kinetics and modulation of glutamate release

Sensory neurons express purinergic P2X receptors on their central and peripheral terminals as well as their cell bodies. ATP activation of these receptors drives action potential firing and glutamate release with potentially important consequences for sensory function. Here we show ATP-gated currents activated in cultured embryonic dorsal root ganglion neurons have heterogeneity of time-courses comparable to those observed in different subpopulations of acutely dissociated adult dorsal root ganglion neurons. The distribution of time-courses across the population of cultured neurons is strongly influenced by culture conditions. Heterogeneity in ATP current kinetics occurs even though immunocytochemical staining reveals a relatively homogeneous and widespread expression of the P2X2 and P2X3 subunits. We show that the time-courses of ATP-gated currents recorded at the cell bodies are mirrored by the time-courses of transmitter release from the dorsal root ganglion nerve terminals, indicating similar P2X receptor properties on the soma and their associated terminals.Our results illustrate a functional heterogeneity of P2X receptor-mediated currents that is strongly influenced by external factors. This heterogeneity in current kinetics may have implications for neuronal function as it constrains the time-course of ATP-mediated modulation of neurotransmitter release at sensory nerve terminals.     

Sphingosine-1-phosphate via activation of a G-protein-coupled receptor(s) enhances the excitability of rat sensory neurons

Sphingosine-1-phosphate (S1P) is released by immune cells and is thought to play a key role in chemotaxis and the onset of the inflammatory response. The question remains whether this lipid mediator also contributes to the enhanced sensitivity of nociceptive neurons that is associated with inflammation. Therefore we examined whether S1P alters the excitability of small diameter, capsaicin-sensitive sensory neurons by measuring action potential (AP) firing and two of the membrane currents critical in regulating the properties of the AP. External application of S1P augments the number of APs evoked by a depolarizing current ramp. The enhanced firing is associated with a decrease in the rheobase and an increase in the resistance at firing threshold although neither the firing threshold nor the resting membrane potential are changed. Treatment with S1P enhanced the tetrodotoxin-resistant sodium current and decreased the total outward potassium current (IK). When sensory neurons were internally perfused with GDP-beta-S, a blocker of G protein activation, the S1P-induced increase in APs was completely blocked and suggests the excitatory actions of S1P are mediated through G-protein-coupled receptors called endothelial differentiation gene or S1PR. In contrast, internal perfusion with GDP-beta-S and S1P increased the number of APs evoked by the current ramp. These results and our finding that the mRNAs for S1PRs are expressed in both the intact dorsal root ganglion and cultures of adult sensory neurons supports the notion that S1P acts on S1PRs linked to G proteins. Together these findings demonstrate that S1P can regulate the excitability of small diameter sensory neurons by acting as an external paracrine-type ligand through activation of G-protein-coupled receptors and thus may contribute to the hypersensitivity during inflammation.     

Localization and function of ATP and GABAA receptors expressed by nociceptors and other postnatal sensory neurons in rat

The role of endogenous GABA and ATP in regulating transmitter release from primary afferent terminals in the superficial dorsal horn of the spinal cord is still controversial. ATP is co-released with GABA from some inhibitory dorsal horn neurons raising the possibility that ATP could act in concert with GABA to regulate transmitter release from primary afferent terminals if receptors to both transmitters are expressed there. Using electrophysiology together with immunocytochemistry, we have investigated the expression of ATP-gated P2X and GABA_A receptors by identified subpopulations of dorsal root ganglion (DRG) neurons known to project primarily to the superficial dorsal horn. Expression of the heat-sensitive vanilloid receptor 1 (VR1) and sensitivity to capsaicin were used to characterize DRG neurons sensitive to noxious heat. Both P2X and GABA_A receptors were expressed on the majority of DRG neurons examined. Recording compound action potentials (CAPs) from dorsal roots in the presence of muscimol, α,β-methylene-ATP (α,β-meATP) or capsaicin resulted in depression of CAP in the slow and medium conducting fibres, indicating cognate receptor expression on the small diameter axons. Dorsal root-evoked dorsal root potentials (DR-DRPs), reflecting depolarization of primary afferent terminals by endogenously released substances, were depressed by the GABA_A receptor antagonist SR95531 and α,β-meATP. These results suggest that GABA_A and P2X receptors are expressed on DRG cell bodies and slow fibre axons, many of which are heat-nociceptive. These fibres project to the superficial lamina of the dorsal horn where the receptors may function to modulate transmitter release near their central terminals.     

Activation of peripheral P2X receptors is sufficient to induce central sensitization in rat medullary dorsal horn nociceptive neurons

Central sensitization and purinergic receptor mechanisms have been implicated as important processes in acute and chronic pain conditions following injury or inflammation of peripheral tissues. This study has documented that application of the P2X(1,2/3,3) receptor agonist αβ-meATP (100mM) to the rat tooth pulp induces central sensitization in medullary dorsal horn nociceptive neurons that is reflected in significant increases in mechanoreceptive field size and responses to noxious stimuli and decreased mechanical activation threshold. Furthermore, these responses can be blocked by pulp application of the P2X(1,2/3,3) antagonist TNP-ATP and also attenuated by medullary application of TNP-ATP. These results suggest that activation of P2X(1,2/3,3) receptors in orofacial tissues plays a critical role in producing central sensitization in medullary dorsal horn nociceptive neurons.     

帕金森病内侧苍白球和丘脑腹外侧核神经细胞放电特点与临床症状的关系

目的：探讨帕金森病（PD）患者内侧苍白球（GPi）和丘脑腹外侧核团（Vop／Vim）细胞电活动与PD症状的关系。方法：24例患者在接受手术的同时采集细胞电活动（GPi：12个,Vop／Vim：12个）和记录肢体肌电图（EMG）。应用单细胞分析,峰阃隔（ISI）、ISI变异系数（CV）和ISI直方图等方法进行分析。用统一帕金森评分量表（UPDRS）进行疗效评估。结果：199个GPi神经元中,33个（16．6％）为与震颤相关放电活动,136个（68．3％）为紧张性放电活动,30个（15．1％）为不规则放电活动。223个Vop／Vim神经元中,110个（49．3％）为与震颤相关的放电活动,49个（22％）为紧张性放电活动,64个（28．7％）为不规则放电活动。ISI分析发现GPi神经元放电频率为78Hz（n=92）而Vop／Vim为24Hz（n=107）。方差分析显示GPi和Vop／Vim的上述三种不同放电模式神经元的数量之间比较差异有统计学意义（P〈0．05）。UPDRS显示,术后GPi对震颤、僵直和运动迟缓的疗效分别为63％、83％和64％;而Vop／Vim术后对震颤、僵直和运动迟缓的疗效分别为94％、66％和49％,提示GPi对僵直改善明显,而Vim对震颤改善显著（P〈0．05）。结论：GPi和Vop／Vim中不同放电模式的神经元可能与PD运动症状有内在联系,支持PD病理生理模型。     

G实验和真菌培养在艾滋病人深部真菌感染中的联合诊断

目的:探讨血清(1-3)-β-D葡聚糖对艾滋病人深部真菌感染的早期诊断。方法:对2014年1月~2014年3月入住我院的68例疑为深部真菌感染的艾滋病人,进行血清(1-3)-β-D葡聚糖检测,并与真菌培养加以对比及结合,观察其在深部真菌感染中的诊断价值。结果:68例疑为深部真菌感染的艾滋病患者,血清(1-3)-β-D葡聚糖阳性率为91.5%,而真菌培养的阳性率仅为57.8%,血清(1-3)-β-D葡聚糖检测阳性率明显高于真菌培养(X2=0.96,P0.05)。血清(1-3)-β-D葡聚糖检测与真菌培养相结合,灵敏度、特异度以及阳性预测值和阴性预测值比单独血清(1-3)-β-D葡聚糖检测或真菌培养阳性率高。结论:血清(1-3)-β-D葡聚糖检测能更早、更灵敏反应深部真菌早期感染;将其与真菌培养结合,能提高深部真菌感染的诊断率。     

Stimulation of mouse cultured sympathetic neurons by uracil but not adenine nucleotides

Cultured neurons from the paravertebral sympathetic chain of rats possess excitatory P2X as well as excitatory uracil nucleotide-sensitive P2Y receptors. Preliminary observations had indicated that the analogous neurons of mice lacked P2X receptors. This difference was now investigated. Thoracolumbar sympathetic neurons from one- to three-day-old mice were cultured for seven days. When the neurons were preincubated with [3H]noradrenaline and then superfused, ATP failed to cause any change in tritium outflow. UTP (3-300 microM) and UDP (30-100 microM), in contrast, caused marked increases, and so did nicotine (3-100 microM). The effect of UTP was not changed by suramin but abolished by tetrodotoxin and in the absence of calcium. The effect of nicotine was antagonized by hexamethonium and also abolished by tetrodotoxin and in the absence of calcium. Pre-exposure to UDP prevented the effect of UTP. In neurons studied by means of whole-cell patch-clamp techniques under current clamp, ATP lacked any effect. UTP (100 microM), UDP (100 microM) and nicotine (10 microM) caused depolarization accompanied by action potentials. Pre-exposure to UDP prevented the effect of UTP. In neurons studied under voltage clamp, ATP, UTP and UDP failed to cause any detectable current. Nicotine (10 microM), in contrast, elicited inward currents. Neither UTP nor UDP reduced the M-type potassium outward current. These results demonstrate a pronounced difference between cultured sympathetic neurons from the mouse and the rat paravertebral chain. Neurons from both species possess the nicotinic acetylcholine receptor. Neurons from both species also possess uracil nucleotide-sensitive P2Y receptors which, when activated, mediate depolarization, action potential firing and noradrenaline release; these effects are not due to inhibition of M-type potassium channels. Only the rat but not the mouse neurons, however, possess P2X receptors which, when activated, mediate cation entry, depolarization, action potential generation and transmitter release. The absence of functional P2X receptors makes the mouse neurons suitable for further study of the uracil nucleotide-sensitive P2Y receptors.     

Effects of gamma-aminobutyric acid on putative sympatho-excitatory neurons in the rat rostral ventrolateral medulla in vitro. Intracellular study

Neurons in the rat rostral ventrolateral medulla (RVLM) were electrophysiologically characterized and identified using an intracellular recording technique in vitro. The recorded neurons could be classified into three types: spontaneously active neurons with a regular pattern of action potential generation; spontaneously active neurons with an irregular pattern of discharge; and silent neurons. In regularly firing neurons during hyperpolarization below spike generation level there occurred: (a) a 'resetting' of regular pattern of firing; (b) the absence of underlying excitatory postsynaptic potentials; (c) an anomalous rectification that produced a decay in the hyperpolarization. In regularly firing neurons, gamma-aminobutyric acid (GABA) (2-5 microM) produced a reversible membrane hyperpolarization, reduction of frequency of discharge and a moderate decrease in membrane input resistance. These effects were completely blocked in the presence of the GABAa antagonists bicuculline (16 microM) or picrotoxin (50 microM). However, the superfusion of bicuculline (16 microM), or picrotoxin (50 microM) alone elicited depolarization, increase in firing rate and increase of membrane input resistance. This study has provided evidence for regularly firing neurons in the RVLM in vitro, with strikingly similar electrophysiological characteristics to a group of neurons described in vivo as tonic sympathoexcitatory. In vitro they are still modulated by gabaergic inputs acting predominantly upon GABAa receptors.     

Purinergic receptor agonists modulate phagocytosis and clearance of apoptotic cells in macrophages

Phagocytosis plays an important role in controlling inflammation and antigen cross-presentation through the uptake of apoptotic bodies from dying cells. As dying cells are known to release nucleotides and other “danger signals”, we investigated whether extracellular nucleotides may affect phagocytosis through binding to P2 purinergic receptors on phagocytic cells. We here show that the purinergic receptor agonists, ATP, ADP, α,β-methylene ATP (α,β-meATP), 3′-O-(4-benzoyl)benzoyl ATP, UTP and UDP, increased phagocytosis of latex beads, and some of them increased endocytosis and/or macropinocytosis of dextran by macrophages. The enhanced phagocytosis could be inhibited by pre-treatment with the P2X and P2Y antagonists, pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid and suramin, and the P2Y₁-selective antagonist, MRS2179. The nucleotides induced upregulation in macrophages of the β2 integrin CD11b/CD18 (Mac-1) and the vitronectin receptor (α_vβ3, CD51/CD61), both of which are involved in recognition and internalization of apoptotic cells. In addition, ATP and α,β-meATP increased adhesion of apoptotic cells to macrophages, both in vitro and in vivo, and α,β-meATP had a small effect on adhesion of necrotic cells. The nucleotides had no effect on adhesion of viable cells. We propose that engagement of the P2 receptors (P2X₁, or P2X₃) by extracellular nucleotides released from dying cells increases the ability of macrophages to bind apoptotic bodies, thus enhancing their ability to internalize and present antigens from the dying cells.