In vivo intrahippocampal microinfusion of carbachol and bicuculline induces theta-like oscillations in the septally deafferented hippocampus.

In their laboratory the authors have previously demonstrated that hippocampal slices could be induced to generate trains of "theta-like" oscillations by whole-bath perfusions of carbachol. Until recently, it has not been possible to generate similar activity in the septally deafferented hippocampus of an otherwise intact brain by microinfusions of carbachol. This study presents a full report of the first demonstration of a theta-like oscillation in the in vivo, septally deafferented hippocampal formation. Rats were anesthetized with urethane and implanted with microinfusion cannulae in the region of the medial septum/vertical limb of the diagonal band of Broca (MS/vDBB) and at single or multiple sites in the stratum moleculare of the fascia dentata. The MS/vDBB was microinfused with procaine hydrochloride to produce a reversible suppression lasting for approximately 20 minutes. Intrahippocampal microinfusions of carbachol or bicuculline alone (in the postprocaine condition of the MS/vDBB) failed to produce any theta-like oscillations. The combination of carbachol and bicuculline produced trains of theta-like oscillations during suppression of the MS/vDBB very similar to those seen in the slice preparations. The oscillations were blocked by intravenous administration of atropine sulfate, and they had the same depth profile as that of theta. Theta-on cells were shown to discharge in rhythmic bursts in synchrony with the oscillations. Thus, it would appear that the essential nature of the medial septal input to the hippocampal formation, for the generation of theta field activity in the intact brain, consists of a critical balance between cholinergic and GABAergic circuitry.     

Early postnatal maturation of GABAA-mediated inhibition in the brainstem respiratory rhythm-generating network of the mouse

It is well established that GABA_A‐mediated postsynaptic potentials are excitatory in many brain regions during embryonic and early postnatal life. The pre‐Bötzinger complex (PBC) in the brainstem is an essential component of the respiratory rhythm‐generating network, where GABA_A‐mediated inhibition plays a critical role in generating a stable respiratory rhythm in adult animals. In the present study, using the perforated patch technique, we investigated the maturation of GABA_A receptor‐mediated effects on rhythmically active PBC neurons and on the motor output in slice preparations from P0–15 neonatal mice. The reversal potential of GABA_A receptor‐mediated current (E_GABA‐A) switched from depolarizing to hyperpolarizing within the first postnatal week. E_GABA‐A was ?13.7 ± 9.8 mV at P0, then it changed to ?44.8 ± 7.0 mV at P2 and ?71.5 ± 6.8 mV at P4. Perfusion of bicarbonate‐free saline has no detectable influence on E_GABA‐A, indicating that a lack of Cl^– extrusion during P0–3 is mainly responsible for early GABA_A‐ergic excitation. At the network level, blockade of GABA_A receptors with bicuculline did not significantly change the frequency of rhythmic bursts recorded from hypoglossal nerve roots before P3, whereas it increased the coefficient of variation. After P3, bicuculline increased burst frequency with little effect on the coefficient of variation. Thus, chloride‐mediated inhibition, which appears in PBC neurons after P3, coincides with the appearance of GABA_A‐mediated modulation of the respiratory rhythm. GABA_A receptor‐activated inhibition may therefore be necessary for frequency modulation in the respiratory network beginning on the fourth postnatal day in the mouse brainstem.     

Topography of dyskinesias and torticollis evoked by inhibition of substantia nigra pars reticulata

GABAergic neurons of the substantia nigra pars reticulata (SNpr) and globus pallidus pars interna (GPi) constitute the output pathways of the basal ganglia. In monkeys, choreiform limb dyskinesias have been described after inhibition of the GPi, but not the SNpr. Given the anatomical and functional similarities between these structures, we hypothesized that choreiform dyskinesias could be evoked by inhibition of an appropriate region within the SNpr. The GABAA receptor agonist, muscimol, was infused into various sites within the SNpr and the adjacent STN of freely moving macaques. The effect of the GABAA antagonist, bicuculline (BIC), was also examined. Muscimol (MUS) in SNpr evoked the following: (1) choreiform dyskinesias of the contralateral arm and/or leg from central and lateral sites; (2) contralaterally directed torticollis from central and posterior sites; and (3) contraversive quadrupedal rotation from anterior and lateral sites. MUS infusions into the adjacent SN pars compacta or STN were without effect, ruling out a contribution of drug spread to adjacent structures. BIC in SNpr induced ipsiversive postures without choreiform dyskinesia or torticollis, whereas in the STN, it evoked ballistic movements. This is the first report of choreiform dyskinesia evoked by inhibition of the SNpr. This highly site‐specific effect was obtained from a restricted region within the SNpr distinct from that responsible for inducing torticollis. These results suggest that overactivity of different SNpr outputs mediates choreiform dyskinesia and torticollis. These abnormalities are symptoms of dystonia, Huntington's disease, and iatrogenic dyskinesias, suggesting that these conditions may result, in part, from a loss of function in SNpr efferent projections. © 2012 Movement Disorder Society     

GABAergic processes in the mesencephalic tegmentum modulate the occurrence of active (rapid eye movement) sleep in guinea pigs

The ventrolateral subdivision of the periaqueductal gray (vlPAG) and the adjacent dorsal mesencephalic reticular formation (dMRF) are involved in the modulation of active (rapid eye movement) sleep (AS). In order to determine the effects on AS of the suppression of neuronal activity in these regions, muscimol, a GABA receptor A (GABA(A)) receptor agonist, and bicuculline, a GABA(A) receptor antagonist, were microinjected bilaterally in guinea pigs and the states of sleep and wakefulness were examined. The main effect of muscimol was an increase in AS; this increase occurred in conjunction with a reduction in the time spent in wakefulness. The powerful effect of muscimol was striking especially when considering the small amount of naturally-occurring AS that is present in this species. Additional observable effects that were induced by muscimol were: 1) long lasting episodes of hypotonia/atonia during wakefulness and quiet sleep that included a lack of extensor tone in the hind limbs, and 2) frequently occurring cortical spindles, similar to those observed during naturally-occurring quiet sleep (sleep spindles), that were present during wakefulness. Conversely, bilateral microinjections of bicuculline induced a prolonged state of wakefulness and blocked the effect of subsequent injections of muscimol. These data suggest that endogenous GABA acts on GABA(A) receptors within the vlPAG and dMRF to promote AS in the guinea pig.     

高效液相色谱法测定夏天无胶囊中原阿片碱、盐酸巴马汀和比枯枯灵碱的含量

目的建立高效液相色谱法测定夏天无胶囊中原阿片碱、盐酸巴马汀、比枯枯灵碱的含量。方法 Agilent ZORBAX SB-C18色谱柱(250 mm×4.6 mm,4.6μm),流动相A为乙腈,流动相B为醋酸(每100 mL 0.2%醋酸用1.5² mL三乙胺调pH=5.1),进行梯度洗脱,流速1.0 mL·min-1,检测波长282 nm,柱温:室温。结果原阿片碱、盐酸巴马汀和比枯枯灵碱分别在4.242 mL三乙胺调pH=5.1),进行梯度洗脱,流速1.0 mL·min-1,检测波长282 nm,柱温:室温。结果原阿片碱、盐酸巴马汀和比枯枯灵碱分别在4.24¹01.8、2.89101.8、2.89⁶9.31和0.4569.31和0.45¹0.84μg·mL-1与峰面积呈良好线性关系,低、中、高3个浓度平均加样回收率均>95%,RSD均<5%。结论该方法准确、快速、专属性强、灵敏度高,可用于夏天无胶囊中原阿片碱、盐酸巴马汀、比枯枯灵碱的含量测定。     

Mechanism of the medium‐duration afterhyperpolarization in rat serotonergic neurons

Most serotonergic neurons display a prominent medium‐duration afterhyperpolarization (mAHP), which is mediated by small‐conductance Ca²⁺‐activated K⁺ (SK) channels. Recent ex vivo and in vivo experiments have suggested that SK channel blockade increases the firing rate and/or bursting in these neurons. The purpose of this study was therefore to characterize the source of Ca²⁺ which activates the mAHP channels in serotonergic neurons. In voltage‐clamp experiments, an outward current was recorded at ?60 mV after a depolarizing pulse to +100 mV. A supramaximal concentration of the SK channel blockers apamin or (‐)‐bicuculline methiodide blocked this outward current. This current was also sensitive to the broad Ca²⁺ channel blocker Co²⁺ and was partially blocked by both ω‐conotoxin and mibefradil, which are blockers of N‐type and T‐type Ca²⁺ channels, respectively. Neither blockers of other voltage‐gated Ca²⁺ channels nor DBHQ, an inhibitor of Ca²⁺‐induced Ca²⁺ release, had any effect on the SK current. In current‐clamp experiments, mAHPs following action potentials were only blocked by ω‐conotoxin and were unaffected by mibefradil. This was observed in slices from both juvenile and adult rats. Finally, when these neurons were induced to fire in an in vivo‐like pacemaker rate, only ω‐conotoxin was able to increase their firing rate (by ~30%), an effect identical to the one previously reported for apamin. Our results demonstrate that N‐type Ca²⁺ channels are the only source of Ca²⁺ which activates the SK channels underlying the mAHP. T‐type Ca²⁺ channels may also activate SK channels under different circumstances.     

基于网络药理学和分子对接技术的抗病毒颗粒治疗新型冠状病毒肺炎（COVID-19）的潜在物质基础研究

目的通过网络药理学及分子对接技术探寻抗病毒颗粒治疗新型冠状病毒肺炎(COVID-19)的潜在物质基础。方法借助TCMSP检索抗病毒颗粒中板蓝根、连翘、石膏、知母、芦根、地黄、广藿香、石菖蒲、郁金的化学成分和作用靶点。通过Uni Prot数据库查询靶点对应的基因,进而运用Cytoscape3.6.1构建药材-化合物-靶点(基因)网络,通过DAVID进行基因本体(GO)功能富集分析和KEGG通路富集分析,预测其作用机制,将药材-化合物-靶点网络中排名前15的成分与新型冠状病毒(SARS-Co V-2)3CL水解酶进行分子对接,同时将比枯枯灵、木犀草素、槲皮素与血管紧张素转化酶Ⅱ(ACE2)进行分子对接。结果药材-化合物-靶点(基因)网络包含药材8个、化合物75个、靶点255个。GO功能富集分析得到GO条目161个(P0.05),其中生物过程(BP)条目65个,细胞组成(CC)条目36个,分子功能(MF)条目60个。KEGG通路富集筛选得到131条信号通路(P0.05)。分子对接结果显示抗病毒颗粒中比枯枯灵、木犀草素、槲皮素等核心活性化合物与SARS-Co V-23CL水解酶的亲和力与临床推荐化学药相似。结论抗病毒颗粒中的活性化合物比枯枯灵、木犀草素、槲皮素等能通过与ACE2结合作用于PTGS2、HSP90AB1、PTGS1等靶点调节多条信号通路,从而可能发挥对COVID-19的治疗作用。     

荷包牡丹碱对海马CA2区的神经毒性与P／Q型钙通道有关

目的：探讨荷包牡丹碱（BiC）对海马CA2区的神经毒性作用以及电压依赖性钙通道（VDCC）对神经毒性作用的影响。方法：BiC6μmol·L^-1刺激培养的大鼠海马组织72h，同时用N，L以及P／Q型钙通道拮抗剂分别阻断相应的钙通道，测定神经细胞摄取的碘化丙啶（PI）。结果：BiC刺激6h后，神经细胞的PI摄取首先出现在CA2区。随着BiC刺激时间的延长，摄取PI的神经细胞的分布范围由CA2区扩散到其他区域。P／Q型VDCC拮抗剂抑制神经细胞的PI摄取，然而N和L型VDCC的拮抗剂无明显抑制效果。结论：在大鼠海马的组织培养中CA2区的神经细胞对BiC的刺激最易受损。CA2区神经细胞的损伤过程中P／Q型VDCC起重要作用。     

Facilitated CA1 hippocampal synaptic plasticity in dystrophin‐deficient mice: Role for GABAA receptors?

Duchenne muscular dystrophy (DMD) is associated with cognitive deficits that may result from a deficiency in the brain isoform of the cytoskeletal membrane-associated protein, dystrophin. CA1 hippocampal short-term potentiation (STP) of synaptic transmission is increased in dystrophin-deficient mdx mice, which has been attributed to a facilitated activation of NMDA receptors. In this study, extracellular recordings in the hippocampal slice preparation were used first to determine the consequences of this alteration on short-term depression (STD). STD induction was facilitated in mdx as compared with wild-type mice in a control medium. Because brain dystrophin deficiency results in a decreased number of gamma-aminobutyric acid A (GABAA)-receptor clusters, we tested the hypothesis that neuronal disinhibition contributes to the enhanced synaptic plasticity in mdx mice. We found that the GABAA receptor antagonist, bicuculline, increased basal neurotransmission in wild-type, but not in mdx mice and prevented the enhanced STP and STD in the CA1 area of slices from mdx mice. The possibility that altered GABA mechanisms underlie the facilitation of NMDA receptor-dependent synaptic plasticity in mdx mice is discussed.     

GABA and bicuculline actions on mouse spinal cord and cortical neurons in cell culture

The neutral amino acid γ-aminobutyric acid (GABA) produced membrane hyperpolarization and increased membrane chloride ion conductance of spinal cord (SC) and cortical (CTX) neurons in cell culture. GABA dose-response curves were obtained for SC neurons by pressure applying known concentrations of GABA from micropipettes with large tips (miniperfusion pipettes). GABA response threshold was about 2 μM and large responses were elicited at GABA concentrations greater than 10 μM. Bicuculline (BICUC) (0.1–10 μM) reversibly antagonized GABA responses on both SC and CTX neurons with a half maximal inhibitory concentration of about 1 μM. BICUC antagonism of GABA responses was competitive (Lineweaver-Burke analysis). These results are compared with data on GABA and BICUC displacement of [³H]GABA binding to membranes of SC and CTX neurons in cell culture. It is suggested that high affinity GABA receptors are likely to be relevant for postsynaptic GABA responses while low affinity GABA receptors may be presynaptic.