Warton S S, Perouansky M, Grantyn R
Department of Neurophysiology, Max Planck Institute for Psychiatry, Martinsried, F.R.G.
Brain Res Dev Brain Res. 1990 Mar 1;52(1-2):95-111. doi: 10.1016/0165-3806(90)90225-n.
Synaptic activity in the superficial (i.e. visual) layer of the superior colliculus was investigated with intracellular microelectrodes using a preparation of the isolated superfused tectum from neonatal rat. It was found that by postnatal day 9 (i.e. before eye opening) the majority of neurons in the superficial gray layer (SGS, stratum griseum superficiale) were already capable of generating Cl(-)-dependent inhibitory postsynaptic potentials (IPSPs) in response to intracollicular stimulation. Properties and development of GABAergic synaptic connections were further characterized in a dissociated cell culture from the SGS. The cultures were prepared from E21 rat embryos and studied between 1 and 38 days in vitro (DIV). gamma-[3H]aminobutyric acid ([3H]GABA) uptake served to identify GABAergic neurons and to estimate their relative density. Axon terminals were labeled by indirect immunostaining for glutamic acid decarboxylase (GAD) and examined with light (LM) and electron microscopy (EM). Responsiveness to exogenous and endogenous GABA was investigated by recording ionic currents with patch clamp techniques. [3H]GABA uptake-positive neurons constituted about 40% of the whole cellular population dissociated from the SGS of E21 rats. After 2 weeks in culture, [3H]GABA uptake was observed in 45-60% of the cells with neuronal features. The relative number of GAD-immunoreactive neuronal perikarya ranged from 28 to 39%, after 2 weeks in vitro. Responsiveness to exogenous GABA was found in all freshly plated neurons. Release of GABA could be demonstrated after 2 DIV by recording spontaneous bicuculline-sensitive Cl- currents. These currents had the characteristics of GABAA receptor-mediated synaptic currents. However, even as late as DIV 6, very few vesicle-containing axonal terminals apposing postsynaptic specializations were revealed with EM. GAD-labeled puncta became clearly visible only after DIV 10-12. Between DIV 14 and 21, the intensity of immunostaining and the density of GAD-labeled synaptic contacts increased, reaching a maximum around DIV 28. GAD-positive puncta covered both neurons and non-neuronal cells. At the level of EM, GAD-positive terminals were shown to establish synaptic contacts with neuronal somata and processes, forming in the majority of cases (22 out of 32 stained terminals) symmetrical contacts. It is concluded that in the SGS of the rat superior colliculus GABAergic neurons and GABAA receptors are present before birth. In dissociated cell cultures ionic currents can be generated in response to endogenous GABA before axonal terminals of GABAergic neurons fully mature. Finally, our experiments show that visual activity is not a prerequisite for the formation of GABAergic synapses between neurons of the SGS.
使用新生大鼠分离的灌流顶盖制备物，通过细胞内微电极研究了上丘表层（即视觉层）的突触活动。研究发现，在出生后第9天（即睁眼之前），表层灰质层（SGS，浅灰质层）中的大多数神经元已经能够在接受丘内刺激时产生Cl(-)依赖性抑制性突触后电位（IPSPs）。在来自SGS的解离细胞培养物中进一步表征了GABA能突触连接的特性和发育。这些培养物由E21大鼠胚胎制备，并在体外培养1至38天（DIV）期间进行研究。γ-[3H]氨基丁酸（[3H]GABA）摄取用于鉴定GABA能神经元并估计其相对密度。轴突终末通过谷氨酸脱羧酶（GAD）的间接免疫染色进行标记，并用光学显微镜（LM）和电子显微镜（EM）检查。通过膜片钳技术记录离子电流来研究对外源性和内源性GABA的反应性。[3H]GABA摄取阳性神经元约占从E21大鼠SGS解离的整个细胞群体的40%。培养2周后，在具有神经元特征的细胞中观察到45 - 60%的细胞有[3H]GABA摄取。体外培养2周后，GAD免疫反应性神经元胞体的相对数量在28%至39%之间。在所有新接种的神经元中都发现了对外源性GABA的反应性。通过记录自发的荷包牡丹碱敏感的Cl-电流，在培养2天后可证明GABA的释放。这些电流具有GABAA受体介导的突触电流的特征。然而，即使在DIV 6时，用EM显示很少有含囊泡的轴突终末与突触后特化结构相对。GAD标记的斑点直到DIV 10 - 12后才清晰可见。在DIV 14至21之间，免疫染色强度和GAD标记的突触接触密度增加，在DIV 28左右达到最大值。GAD阳性斑点覆盖神经元和非神经元细胞。在EM水平上，GAD阳性终末显示与神经元胞体和突起建立突触接触，在大多数情况下（32个染色终末中的22个）形成对称接触。得出的结论是，在大鼠上丘的SGS中，GABA能神经元和GABAA受体在出生前就已存在。在解离细胞培养物中，在GABA能神经元的轴突终末完全成熟之前，可对内源性GABA产生离子电流反应。最后，我们的实验表明，视觉活动不是SGS神经元之间形成GABA能突触的先决条件。
