Whole-cell recording of the robust nucleus of the arcopallium neurons in the adult zebra finch

BACKGROUND： Electrophysiological properties of the song nucleus have been revealed using conventional techniques, such as intracellular and extracellular recording. Research concerning the neuronal activation properties and regulations of the song system at the cellular and ion channel level may help reveal the neural mechanism of song learning. OBJECTIVE： To perform whole-cell recording of robust nucleus of the arcopallium （RA） neurons in brain slices from adult zebra finches （Taeniopygia guttata） and observe the action potential, sodium/potassium current and the spontaneous postsynaptic current of RA neurons. DESIGN, TIME AND SETTING： Self-controlled, neuroelectrophysiological experiment. The study was performed at the Neurophysiology Laboratory of South China Normal University from April to September 2008. MATERIALS： Flaming/Brown puller P-97 was purchased from Sutter Ins, USA; Axopatch 700B amplifier and Digidata 1332A converter were purchased from Axon Instrument, USA; pClamp software was provided by Axon Instrument, USA. METHODS： RA neurons were acutely isolated from 24 healthy male zebra finches. The action potential, voltage-gate sodium/potassium current and spontaneous postsynaptic current were recorded by whole-cell recording technology. Data were analyzed by pClamp software. MAIN OUTCOME MEASURES： The amplitude and frequency of the action potential, and the amplitude of the voltage-dependent and spontaneous postsynaptic currents, were measured. RESULTS： （1） Testing of action potential： Cells exhibited a stable current-voltage relationship following a series of hyperpolarization stepped currents, and an action potential was triggered by the spike threshold. All the recorded cells displayed repetitive firing following depolarizing current injection, with a frequency beyond 100 Hz. （2） Testing of voltage-gate currents： The inward and outward whole-cell currents were observed after a series of depolarizing voltage steps. The inward current disappeared following the application of tetrodotoxin and the outward current was significantly inhibited by application of 4-aminopyfidione and tetraethylammonium chloride. （3） Testing of spontaneous postsynaptic current： The majority of recorded cells exhibited an inward synaptic current when the membrane potential was maintained at -60 mV, with some cells exhibiting a robustly outward current when the membrane potential was maintained at -30 mV. Tetrodotoxin was unable to affect the spontaneous postsynaptic current. Following application of bicuculline [y-aminobutyric acid （A） receptor antagonist] and high concentration kynurenic acid （ionotropic glutamate receptor antagonist）, the inward and outward currents were completely inhibited. CONCLUSION： Under these experimental conditions, the action potential, sodium/potassium current and spontaneous postsynaptic current were recorded successfully in RA neurons. This indicates that the cells preserved relatively intact synaptic connections and normal physiological activity, which is required for investigating ion channels. The inward and outward whole-cell currents were sodium and potassium currents, respectively. The postsynaptic y-aminobutyric acid （A） receptors and ionotropic glutamate receptors contributed to the spontaneous postsynaptic current.

Sexual dimorphism of the electrophysiological properties of the projection neurons in the robust nucleus of the arcopallium in adult zebra finches

Objective To observe the sexual differences in electrophysiological properties of neurons in the robust nucleus of the arcopallium （RA） in adult zebra finches, and to provide the direct electrophysiological evidence for the sexual dimorphism of birdsong. Methods Whole-cell recording was used to record the spontaneous action potential firing rates from RA projection neurons in acute brain slices. Results The projection neurons of RA in male birds fired spontaneously at 10 Hz or above, while in female birds, the frequency was significantly lower, and even no firings could be detected. Conclusion There is a sexual difference in electrophysiological properties of projection neurons in RA, which may result from the difference in the levels of steroid hormones in birds.

SK channels modulate the excitability and firing precision of projection neurons in the robust nucleus of the arcopallium in adult male zebra finches

Objective Motor control is encoded by neuronal activity. Small conductance Ca2＋-activated K＋ channels （SK channels） maintain the regularity and precision of firing by contributing to the afterhyperpolarization （AHP） of the action potential in mammals. However, it is not clear how SK channels regulate the output of the vocal motor system in songbirds. The premotor robust nucleus of the arcopallium （RA） in the zebra finch is responsible for the output of song information. The temporal pattern of spike bursts in RA projection neurons is associated with the timing of the acoustic features of birdsong. Methods The firing properties of RA projection neurons were analyzed using patch clamp whole-cell and cell-attached recording techniques. Results SK channel blockade by apamin decreased the AHP amplitude and increased the evoked firing rate in RA projection neurons. It also caused reductions in the regularity and precision of firing. RA projection neurons displayed regular spontaneous action potentials, while apamin caused irregular spontaneous firing but had no effect on the firing rate. In the absence of synaptic inputs, RA projection neurons still had spontaneous firing, and apamin had an evident effect on the firing rate, but caused no significant change in the firing regularity, compared with apamin application in the presence of synaptic inputs. Conclusion SK channels contribute to the maintenance of firing regularity in RA projection neurons which requires synaptic activity, and consequently ensures the precision of song encoding.

黄雀端脑古纹状体栎核的纤维联系──HRP法研究

用ＨＲＰ顺、逆行追踪法，研究黄雀（Ｃａｒｄｕｅｌｉｓｓｐｉｎｕｓ）古纹状体栎（粗）核（ＲＡ）的中枢联系。将ＨＲＰ以微电泳导入ＲＡ后，在同侧的中脑背内侧核、舌下神经核气管鸣管部及枕中脑束见到密集的顺行标记终末或纤维；在同侧的上纹状体腹侧尾核、新纹状体前部大细胞核外侧部、古纹状体带核及脑桥蓝斑内出现了大量逆行标记细胞。结果显示，ＲＡ是端脑内发声控制的重要运动中枢，它可能受上纹状体腹侧尾核、新纹状体前部大细胞核外侧部、古纹状体带核及脑桥蓝斑的调控。

燕雀前脑古纹状体粗核超微结构的观察

实验用鸟经脑灌流后于RA取材 ,用Epon 81 2包埋剂浸润包埋 ,经铀 -铅电子染色后 ,H -60 0投射电镜下观察 ,IBAS图像分析仪进行处理 .结果发现 ,燕雀RA的轴 -体突触较多见 .突触类型包括GrayⅠ型和GrayⅡ型 .Ⅰ型突触后膜显著增厚 ,突触前含有清亮圆形小泡 ;Ⅱ型突触前后膜对称 ,突触小泡多为扁平囊泡 .经统计学t检验 ,突触总数、GrayⅠ型突触数和棘突触数 ,雄鸟多于雌鸟 ,性别差异极显著 (P <0 .0 1 ) .

黑尾蜡嘴雀原纹状体发声控制核团的传入性纤维联系

将HRP微电泳人黑尾蜡嘴雀(Eophona mihratoria)端脑原纹状体粗核内,观察其传入性纤维联系.在下列部位发现有密集的逆行标记细胞:(1):原纹状体带核;(2)上纹状体腹侧尾核;(3)新纹状体前部大细胞核外侧部;(4)脑桥蓝斑.以上标记细胞均出现在同侧.结果表明,原纹状体粗核接受原纹状体带核、上纹状体腹侧尾核、新纹状体前部大细胞核外侧部及脑桥蓝斑的传入性投射.

白腰文鸟原纹状体栎核发声通路与SP-免疫组化的定位

目的 :对白腰文鸟端脑原纹状体栎核 (RA)的神经联系和SP 免疫阳性神经细胞或纤维在发声通路中的定位进行研究。方法 :采用生物素结合的葡聚糖胺 (BDA)双向示踪方法和P物质 (substanceP ,SP)免疫组织化学技术。将BDA微量注入RA内 ,经ABC反应。结果 :①RA接受发声控制的高位中枢HVC和新纹状体前部巨细胞核 (MAN)外侧部的传入投射 ,由RA发出的神经纤维投射到中脑背内侧核 (DM )和延髓舌下神经核气管鸣管部(nXIIts) ,由此组成发声控制通路。②SP 免疫阳性神经细胞大量分布在端脑的HVC ,MAN ,RA内。SP 免疫阳性神经纤维或末梢密集分布在发声运动中枢中脑的DM、延髓的nXIIts、舌下神经 ,以及中脑背外侧核壳区 (MLdshell)、中脑丘间核 (ICo)和丘脑卵圆核壳 (OVshell)内。 结论 :由于SP广泛分布在发声控制中枢内 ,提示SP可能在发声控制方面具有重要的生理功能。

金丝雀发声和其它功能核团向原纹状体栎核的投射

用ＨＲＰ示踪方法研究金丝雀端脑原纹状体栎核的传入和传出投射。将ＨＲＰ微电泳入该核内，观察其顺、逆行标记物的分布，结果表明，原纹状体栎核接受上纹状体腹侧尾核、新纹状体前部巨细胞核外侧部、原纹状体带核及蓝斑核的传入投射，由原纹状体栎核发出的纤维支配中脑丘间核背内侧部和延髓舌下神经核气管鸣管部．因此，原纹状体栎核不仅是端脑内发声控制的主要运动中枢，它可能还司其它功能。