Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank''s Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1°C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.     

小鼠初级听皮质神经元的强度调谐特性与机制分析

强度是声音的基本参数之一,听神经元的强度调谐在听觉信息处理方面具有重要意义．以往研究发现γ-氨基丁酸(γ-aminobutyric acid, GABA)能抑制性输入在强度调谐的形成过程中起重要作用,但对抑制性输入与局部神经回路之间的关系并不清楚．本实验通过在体细胞外电生理记录和神经药理学方法,分析了小鼠初级听皮质神经元的强度调谐特性,结果显示：单调型神经元在声刺激强度自中等强度增高时潜伏期缩短(P < 0.05)且发放持续时间延长(P < 0.05),非单调型神经元在声刺激强度自最佳强度增高时潜伏期不变且发放持续时间缩短(P < 0.01)．注射GABA能阻断剂荷包牡丹碱(bicuculline, Bic)后,39.3%的神经元强度调谐类型不变,42.9%的神经元非单调性减弱,17.9%的神经元非单调性增强．表明GABA能抑制并非是形成非单调性的唯一因素,兴奋性输入本身的非单调性和高阈值非GABA能抑制的激活也可能在其中发挥作用．推测由兴奋性和抑制性输入所构成的局部神经功能回路及其整合决定了听皮质神经元的强度调谐特性．     

猫纹状皮层神经元整合野的形状和范围

用正弦调制的移动光栅测量了１２０个猫纹状皮层神经元在长度和宽度方向上的空间整合特性。结果表明：（１）大多数细胞的传统感受野具有长宽相近的结构,然而它们的整合野多数是宽而短或者窄而长的长条形。（２）整合野大小为传统感受野的２—７倍,平均为３．７倍。简单细胞与复杂细胞整合野的大小没有显著差别。（３）随着感受野的视网膜偏心度的增加,整合野大小有逐渐增大的趋势。（４）感受野靠近视网膜垂直中线的细胞,其整合野可以跨越中线进入同侧视野;在两半视野中整合野的范围没有显著差异,但是同侧视野整合野的作用强度明显弱于对侧视野。以上结果提示,初级视皮层神经元能够对大范围内的图形特征进行整合,这种整合作用来自两侧视野。     

Early network activity propagates bidirectionally between hippocampus and cortex

Spontaneous activity in the developing brain helps refine neuronal connections before the arrival of sensory‐driven neuronal activity. In mouse neocortex during the first postnatal week, waves of spontaneous activity originating from pacemaker regions in the septal nucleus and piriform cortex propagate through the neocortex. Using high‐speed Ca²⁺ imaging to resolve the spatiotemporal dynamics of wave propagation in parasagittal mouse brain slices, we show that the hippocampus can act as an additional source of neocortical waves. Some waves that originate in the hippocampus remain restricted to that structure, while others pause at the hippocampus‐neocortex boundary and then propagate into the neocortex. Blocking GABAergic neurotransmission decreases the likelihood of wave propagation into neocortex, whereas blocking glutamatergic neurotransmission eliminates spontaneous and evoked hippocampal waves. A subset of hippocampal and cortical waves trigger Ca²⁺ waves in astrocytic networks after a brief delay. Hippocampal waves accompanied by Ca²⁺ elevation in astrocytes are more likely to propagate into the neocortex. Finally, we show that two structures in our preparation that initiate waves—the hippocampus and the piriform cortex—can be electrically stimulated to initiate propagating waves at lower thresholds than the neocortex, indicating that the intrinsic circuit properties of those regions are responsible for their pacemaker function. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 661–672, 2016     

An Excitatory Neural Assembly Encodes Short-Term Memory in the Prefrontal Cortex

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Kinetics of tert-[35S]Butylbicyclophosphorothionate Binding in the Cerebral Cortex of Newborn and Adult Rats: Effects of GABA and Receptor Desensitization

Abstract: The effects of GABA on the kinetics of tert -[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS) binding to the convulsant site of GABA_A receptors were studied in membrane suspensions from the cerebral cortex of newborn (1-day-old) and adult (90-day-old) rats. TBPS dissociation was biphasic in neonates and adults, indicating that more than one interconvertible state of [³⁵S]TBPS binding sites may be present in the cerebral cortex. In the absence of GABA, the fast ( t _1/2, 11 min) and slow ( t _1/2, 77 min) components of TBPS dissociation in newborn rats were approximately fourfold slower than in adults. The acceleration of the dissociation rates caused by 2 µ M GABA, however, was more robust in neonates than in adults (six- to ninefold vs. twofold increase, respectively). Moreover, the dissociation rates of TBPS in membranes preincubated with 2 µ M GABA (dissociation started by adding 40 µ M picrotoxin) were two- to fourfold slower than in membranes preincubated without GABA (dissociation started by adding 40 µ M picrotoxin plus 2 µ M GABA). Taken together, these results suggest that (1) the closed state of GABA_A receptors is associated with a more effective steric barrier for the binding of TBPS in neonates compared with adults, (2) GABA produces a larger acceleration of the binding kinetics of TBPS in neonates than in adults, and (3) long incubations with GABA may cause receptor desensitization, which in turn slows down the dissociation rates of TBPS.     

Effects of endogenous and exogenous cholesterol on the ultrastructure and steroid secretion of undifferentiated rat adrenocortical cells in primary culture

Summary The present study was undertaken to define the effects of lipoprotein-derived cholesterol and endogenous, de novo synthesized cholesterol on the ultrastructure and function of undifferentiated rat adrenocortical cells [lipoprotein (HDL₃ and LDL) receptor-negative, zona glomerulosa-like adrenocortical cells] in primary culture. For this purpose human plasma high density lipoprotein (HDL₃) or low density lipoprotein (LDL) was added to culture medium devoid of cholesterol. Steroid secretion remained at the low basal level even after addition of lipoproteins, and the amount of intracellular lipid droplets did not increase. When mevinolin (0.96 µg/ml), an inhibitor of cholesterol synthesis, was added to the culture medium, a low secretion of corticosterone was measured both in serum-free and serum-containing media. Ultrastructurally, lipid droplets disappeared after treatment with mevinolin in both media used. At this concentration of mevinolin cell proliferation was similar to that in the controls, but at higher concentrations (4.8 or 9.6 µg/ml) proliferation was inhibited to 42% and 26% in serum-free medium, and 20% and 12% in serum-supplemented medium, respectively. This study demonstrates that cell proliferation and synthesis of corticosterone by undifferentiated rat adrenocortical cells is identical in the absence or presence of exogenous lipoprotein cholesterol. Inhibition of de novo cholesterol synthesis by mevinolin over a period of 7 days does not inhibit corticosterone secretion or proliferation of cells but decreases the amount of intracellular lipid droplets, thus suggesting utilization of intracellular cholesterol esters. However, higher concentrations of mevinolin inhibit proliferation of cells both in serum-free and serum-containing media.     

右美托咪定对腰椎全麻手术患者术后疼痛及认知功能的影响

目的:探讨右美托咪定对腰椎全麻手术患者术后疼痛及认知功能的影响。方法:选择2014年3月~2015年12月在我院行腰椎全麻手术的84例患者为研究对象,按照随机数字表法分为对照组(42例)和试验组(42例),患者均常规给予芬太尼及顺式阿曲库铵麻醉诱导,试验组患者在麻醉诱导过程中给予右美托咪定静脉注射,对照组患者仅给予氯化钠注射液静脉注射。分别于术前(T0)、手术开始2 h(T2)、术后24 h(T24)检测血清肾上腺糖皮质激素,采用疼痛视觉模拟评分法(VAS)进行疼痛评定;采用简易智能精神状态量表(MMSE)于术后1d和2d进行认知状态评定,并计算术后认知功能障碍(POCD)发生率;同时观察患者不良反应发生情况。结果:试验组患者T2和T24时肾上腺糖皮质激素水平明显低于T0,T2时试验组患者肾上腺糖皮质激素水平明显低于对照组,差异有统计学意义(P0.05)。试验组患者T2时VAS评分明显低于对照组,差异有统计学意义(P0.05)。术后1d和2d时试验组患者的MMSE评分高于对照组,POCD发生率明显低于对照组;两组患者术后2d时MMSE评分高于术后1d,POCD发生率明显低于术后1d,差异均有统计学意义(P0.05)。两组患者均未见除POCD以外的不良反应。结论:右美托咪定有较强的抗氧化能力,可有效减轻腰椎全麻手术患者的疼痛程度,提高患者的认知功能。     

经验改变大鼠听皮层神经元的特征频率

应用常规电生理学技术,以神经元的特征频率和频率调谐曲线为指标,研究大鼠听皮层神经元特征频率的可塑性. 结果表明,在给予的条件刺激频率和神经元特征频率相差1.0 kHz范围内,条件刺激可诱导50%以上神经元特征频率发生完全偏移,并可分为向频率调谐曲线的低频端偏移、高频端偏移,或两侧均可偏移三种类型. 其中,神经元的特征频率高、Q₁₀-dB值大和频率调谐曲线对称指数大于零的神经元,其特征频率偏向频率调谐曲线高频端的概率更高. 结果提示,经验可改变大鼠听皮层神经元的特征频率,为深入研究中枢神经元功能活动可塑性的机制提供了重要实验资料.