Department of Biological Structure, University of Washington, 1950 Pacific Ave, Seattle, WA 98195, USA.
Neural Dev. 2011 Aug 24;6:31. doi: 10.1186/1749-8104-6-31.
Neuronal output is shaped by a balance of excitation and inhibition. How this balance is attained in the central nervous system during development is not well understood, and is complicated by the fact that, in vivo, GABAergic and glycinergic synaptogenesis precedes that of glutamatergic synapses. Here, we determined the distributions of inhibitory postsynaptic sites on the dendritic arbors of individual neurons, and compared their developmental patterns with that of excitatory postsynaptic sites. We focused on retinal ganglion cells (RGCs), the output neurons of the retina, which receive excitatory input from bipolar cells and inhibitory input from amacrine cells. To visualize and map inhibitory postsynaptic sites, we generated transgenic mice in which RGCs express fluorescently tagged Neuroligin 2 (YFP-NL2) under the control of the Thy1 promoter. By labeling RGC dendrites biolistically in YFP-NL2-expressing retinas, we were able to map the spatial distribution and thus densities of inhibitory postsynaptic sites on the dendritic arbors of individual large-field RGCs across ages.
We demonstrate that YFP-NL2 is present at inhibitory synapses in the inner plexiform layer by its co-localization with gephyrin, the γ2 subunit of the GABAA receptor and glycine receptors. YFP-NL2 puncta were apposed to the vesicular inhibitory transmitter transporter VGAT but not to CtBP2, a marker of presynaptic ribbons found at bipolar cell terminals. Similar patterns of co-localization with synaptic markers were observed for endogenous NL2. We also verified that expression of YFP-NL2 in the transgenic line did not significantly alter spontaneous inhibitory synaptic transmission onto RGCs. Using these mice, we found that, on average, the density of inhibitory synapses on individual arbors increased gradually until eye opening (postnatal day 15). A small centro-peripheral gradient in density found in mature arbors was apparent at the earliest age we examined (postnatal day 8). Unexpectedly, the adult ratio of inhibitory/excitatory postsynaptic sites was rapidly attained, shortly after glutamatergic synaptogenesis commenced (postnatal day 7).
Our observations suggest that bipolar and amacrine cell synaptogenesis onto RGCs appear coordinated to rapidly attain a balanced ratio of excitatory and inhibitory synapse densities prior to the onset of visual experience.
神经元的输出由兴奋和抑制的平衡来塑造。在中枢神经系统中，这种平衡是如何在发育过程中实现的，目前还不是很清楚，而且由于 GABA 能和甘氨酸能突触的形成先于谷氨酸能突触，情况变得更加复杂。在这里，我们确定了单个神经元树突上抑制性突触后位点的分布，并将其发育模式与兴奋性突触后位点的发育模式进行了比较。我们专注于视网膜神经节细胞（RGC），它们是视网膜的输出神经元，从双极细胞接收兴奋性输入，从无长突细胞接收抑制性输入。为了可视化和映射抑制性突触后位点，我们在 Thy1 启动子的控制下，使 RGC 表达荧光标记的神经连接蛋白 2（YFP-NL2），生成了转基因小鼠。通过在 YFP-NL2 表达的视网膜中生物弹击标记 RGC 树突，我们能够绘制单个大视野 RGC 树突上抑制性突触后位点的空间分布，从而确定其密度。
我们通过 YFP-NL2 与 GABAA 受体 γ2 亚基和甘氨酸受体的共定位证明 YFP-NL2 存在于内丛状层的抑制性突触中。YFP-NL2 斑点与囊泡抑制性递质转运体 VGAT 共定位，但与 CtBP2 （双极细胞末端的突触前带的标志物）不共定位。内源性 NL2 也观察到类似的与突触标志物共定位的模式。我们还验证了转基因组中 YFP-NL2 的表达并未显著改变 RGC 上的自发性抑制性突触传递。使用这些小鼠，我们发现，平均而言，单个树突上抑制性突触的密度逐渐增加，直到睁眼（出生后 15 天）。在我们研究的最早年龄（出生后 8 天）就可以发现成熟树突上存在小的中心-外周密度梯度。出乎意料的是，谷氨酸能突触形成开始后（出生后 7 天），抑制性/兴奋性突触后位点的成年比值很快就达到了。
我们的观察结果表明，双极细胞和无长突细胞与 RGC 的突触形成似乎协调一致，在视觉体验开始之前迅速达到兴奋性和抑制性突触密度的平衡比值。
