Mechanisms Of Synaptic Plasticity In The Adult And Developing Nervous System

环境健康的一个长期问题是需要了解环境在人类大脑发育中的作用。新生儿的大脑特别容易受到感官环境的破坏，这可能对其生理和形态产生深远的影响。发育中的大脑对感官操纵的环境影响或对环境毒物的敏感性在出生后生命的限定关键时期特别明显。一方面，这种易感性使发育中的大脑特别容易受到有毒物质的伤害。另一方面，神经元或突触之间连接的可塑性对于在出生后发育期间改善大脑回路至关重要。改变突触的类似机制可能是成人学习的基础。因此，我们的主要兴趣是确定持久突触可塑性的分子基础。为了了解神经元活动如何诱导神经元的持久修饰，我们使用了分子，生物化学，电生理和成像技术的各种集合。我们主要使用新生和成年大鼠和小鼠的海马切片制备。海马体相对简单的层状结构，本身在学习和记忆中起着重要作用，使电生理学研究很容易进行。 为了测量突触反应，我们使用的技术，包括全细胞膜片钳记录保持在体外切片和场电位记录从急性准备海马切片。 在多电极阵列上生长的切片培养物允许在双光子共聚焦荧光显微镜期间进行细胞外刺激和记录。为了确定神经元活动如何调节转录，我们将联合收割机分子和生物化学方法与海马切片的电刺激相结合。 急性分离的海马和皮质神经元细胞培养物，可以刺激神经元以模拟LTP和LTD，也可用于生化研究和荧光成像实验。 为了了解突触变化如何持续一生，我们研究神经元活动如何调节基因转录以巩固突触变化。 有证据表明，突触功效的长期变化需要新RNA的表达，为此，我们专注于神经元动作电位对基因转录的调节。以前，我们已经表明，动作电位产生的某些频率的突触刺激（5和100赫兹）是更敏感的NMDA受体阻滞剂比那些诱导的θ-突发模式的刺激。 这种敏感性的差异解释了激酶激活如何被阻断动作电位的相同浓度的NMDA受体抑制剂在5和100 Hz的情况下阻断，而不是theta-爆发刺激，如通过对磷酸化并因此活化的细胞外信号调节激酶（ERK）的抗体染色所评估的。 如果用抑制性突触的阻滞剂恢复动作电位，染色可以被挽救。 我们现在已经发现了类似的结果与激活的几个转录因子和转录的活性调节基因，弧/arg3.1（诱导是NMDA受体的独立性，只要动作电位被保存）。这些发现对于解释使用NMDA受体抑制剂的实验具有重要意义，从而得出结论，即细胞核的信号来自突触。这些结果支持我们的想法，动作电位是至关重要的一些基因在生理条件下的转录，并将导致更好地了解所需的基因突触可塑性的巩固是如何调节。 ?通过比较高度可塑性的大脑区域（如海马体的CA 1区）和可塑性较低的区域（如大脑皮层的第4层），可能会对突触可塑性有一些了解。海马体对记忆和空间导航至关重要。然而，海马体的一个区域，CA 2，与第4层的几个基因（TREK-1，钾通道，例如）的表达相同，因此我们预测它将共享第4层神经元的特征，例如对突触可塑性的抵抗。有趣的是，CA 2因其对创伤、缺血和中风引起的疾病和损伤的抵抗力而闻名。正如预测的那样，我们发现CA 2对突触可塑性的形式具有类似的抵抗力，包括突触加强（长时程增强，LTP）和突触减弱（长时程抑制，LTD），即使CA 2中的突触反应与邻近的CA 1和CA 3区域中的突触反应非常相似。由于CA 2及其周围区域在解剖学上非常相似，因此这些发现可能导致识别不仅导致突触可塑性，而且导致神经元损伤和死亡的途径中的关键分子组分。 利用我们从CA 2中学到的信息，我们的目标是确定突触可塑性在关键时期的形式的发育下调的性质。我们的长期目标是确定神经元活动如何导致突触消除（修剪）。我们现在已经开发出一种技术，通过这种技术，可以在活组织中研究关键时期的活动依赖性突触消除。这种方法将使我们能够测试我们关于LTD如何导致突触丢失的想法。 通过了解发育过程中突触可塑性的分子和细胞机制，我们可能开始了解发育过程中暴露于环境毒物如何对认知和疾病易感性产生终身影响。

A longstanding issue in environmental health is the need to understand the role the environment plays in human brain development. The brain of the neonate is particularly susceptible to disruption of the sensory environment, which can have profound effects on its physiology and morphology. Such susceptibility of the developing brain to environmental influence by sensory manipulation or to environmental toxicants is particularly pronounced during defined critical periods of postnatal life. On the one hand, this susceptibility makes the developing brain particularly vulnerable to toxic insults. On the other hand, the plasticity of the connections between neurons, or synapses, is critical for refining brain circuitry during postnatal development. Similar mechanisms for changing synapses are likely to serve the basis for learning in the adult. Our primary interest, therefore, has been to determine the molecular basis of long-lasting synaptic plasticity. Toward our goal of learning how neuronal activity can induce lasting modifications in neurons, we use a diverse collection of molecular, biochemical, electrophysiological, and imaging techniques. We primarily use the hippocampal slice preparation using neonate and adult rats and mice. The relatively simple laminar structure of the hippocampus, which itself plays an important role in learning and memory, allows electrophysiological studies to be performed easily. To measure synaptic responses, we use techniques that include whole-cell patch clamp recordings from slices maintained in vitro and field potential recordings from acutely prepared hippocampal slices. Slice-cultures grown on multielectrode arrays allow for extracellular stimulating and recording during two-photon confocal fluorescent microscopy. To determine how transcription is regulated by neuronal activity, we combine molecular and biochemical methods with electrical stimulation of hippocampal slices. Acutely dissociated hippocampal and cortical neuronal cell cultures, which can be stimulated pharmacologically to mimic LTP and LTD, are also used for both biochemical studies and fluorescent imaging experiments. To understand how synaptic changes persist for up to a lifetime, we study how neuronal activity regulates gene transcription to consolidate synaptic changes. Evidence suggests that the long-term changes in synaptic efficacy require expression of new RNA and toward that end, we have focused on the regulation of gene transcription by neuronal action potentials. Previously, we have shown that action potentials generated with certain frequencies of synaptic stimulation (5 and 100 Hz) are more sensitive to NMDA receptor blockers than those induced with a theta-burst pattern of stimulation. This difference in sensitivity explained how kinase activation, as assessed by staining for an antibody against the phosphorylated and therefore activated extracellular signal-regulated kinase (ERK), is blocked in the 5 and 100 Hz cases, but not the theta-burst stimulation, by the same concentrations of NMDA receptor inhibitors that block the action potentials. The staining could be rescued if action potentials are restored with a blocker of inhibitory synapses. We have now found similar results with the activation of several transcription factors and transcription of an activity-regulated gene, arc/arg3.1 (induction was NMDA receptor independent, provided that action potentials were preserved). These findings have important implications for the interpretation of experiments using NMDA receptor inhibitors to conclude that signals to the nucleus come from the synapse. These results support our idea that action potentials are critical to the transcription of some genes under physiological conditions and will lead to a better understanding of how genes required for the consolidation of synaptic plasticity are regulated. ?Some insights into synaptic plasticity might be gained by comparing highly plastic brain areas, such as the CA1 area of hippocampus, with less plastic areas, such as layer 4 of the cerebral cortex. The hippocampus is critical for memory and spatial navigation. One area of the hippocampus, the CA2, however, shares with layer 4 expression of several of genes (TREK-1, a potassium channel, for example), and so we predicted that it would share features of layer 4 neurons such as its resistance to synaptic plasticity. Interestingly, the CA2 has been noted for its resistance to disease and damage from trauma, ischemia, and stroke. As predicted, we found that CA2 is similarly resistant to forms of synaptic plasticity including synapse strengthening (long-term potentiation, LTP) and synaptic weakening (long-term depression, LTD), even though synaptic responses in CA2 were very similar to those in the neighboring CA1 and CA3 areas. Because CA2 and its surrounding regions are anatomically very similar, these findings may therefore lead to identification of critical molecular components in the pathways leading to not only synaptic plasticity, but also neuronal damage and death. Using information we learn from CA2, we aim to determine the nature of the developmental down-regulation of synaptic plasticity in the form of critical periods. Our longer term goal is to determine how neuronal activity leads to synapse elimination (pruning). We have now developed a technique by which activity-dependent synapse elimination during critical periods can be studied in live tissue. This method will allow us to test our ideas on how LTD might lead to synaptic loss. By understanding the molecular and cellular mechanisms of synaptic plasticity during development, we may begin to understand how exposure to environmental toxicants during development can have life-long consequences on cognition and susceptibility to disease.