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所起的作用。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或环境毒物的环境影响的这种易感性在出生后生命的特定关键时期尤为明显。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，对于在出生后发育过程中完善大脑回路至关重要。类似的突触变化机制可能是成人学习的基础。因此，我们的主要兴趣是确定突触长期可塑性的分子基础。为了实现我们学习神经元活动如何诱导神经元持久变化的目标，我们使用了多种分子、生化、电生理和成像技术。我们主要使用新生和成年大鼠和小鼠的海马切片制备。海马相对简单的层状结构本身在学习和记忆中起着重要作用，这使得电生理研究很容易进行。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和体外保存的急性制备海马切片的场电位记录。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下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，我们使用分子和生化方法对急性分离的海马和皮质神经元细胞进行培养，这些细胞可以通过药理学刺激来模拟LTP和LTD。为了了解突触变化如何持续一生，我们研究了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，突触效能的长期变化需要新RNA的表达，为此，我们重点研究了神经元动作电位对基因转录的调节。在此之前，我们已经证明，在特定频率的突触刺激（5和100赫兹）下产生的动作电位对NMDA受体阻滞剂的敏感性要高于在脉冲刺激模式下产生的动作电位。这种敏感性的差异解释了激酶激活是如何通过对磷酸化并因此激活的细胞外信号调节激酶（ERK）的抗体染色来评估的，在5和100 Hz的情况下，通过相同浓度的阻断动作电位的NMDA受体抑制剂来阻断，而不是阻断theta-burst刺激。如果使用抑制性突触阻滞剂恢复动作电位，则可以挽救染色。我们现在已经发现了几个转录因子的激活和一个活性调节基因arc/arg3.1的转录的类似结果（诱导是NMDA受体独立的，只要动作电位被保留）。这些发现对使用NMDA受体抑制剂的实验的解释具有重要意义，该实验得出了传递给细胞核的信号来自突触的结论。这些结果支持了我们的观点，即动作电位在生理条件下对某些基因的转录至关重要，并将使我们更好地理解突触可塑性巩固所需的基因是如何被调节的。

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 and field potential recordings from acutely prepared hippocampal slices maintained in vitro. 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 use molecular and biochemical methods with acutely dissociated hippocampal and cortical neuronal cell cultures, which can be stimulated pharmacologically to mimic LTP and LTD. 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 hypothesized that it would share with layer 4 neurons features such as a resistance to synaptic plasticity. The CA2, incidentally, has been noted for its resistance to disease and damage from trauma, ischemia, and stroke. As predicted, we discovered 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 LTP requires postsynaptic calcium for its induction, a first step in assessing how CA2 differs from its neighboring subfields was to test whether comparable levels of free calcium could be achieved in CA2 neurons in response to action potential generation. Using 2-photon laser scanning microscopy and whole-cell recordings of hippocampal neurons in slices, we found that dendritic spines in CA2 have very different calcium dynamics from spines in CA1 and CA3. Both calcium buffering capacity and rates of calcium extrusion were higher in CA2 spines when compared with those in the neighboring regions. When calcium extrusion was disrupted by inhibition of the plasma membrane calcium ATPase, LTP was restored. These data indicate that in spite of the multitude of other potential negative modulators of plasticity concentrated in CA2, blockade of calcium extrusion alone was sufficient to restore it. 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. We found that electrical stimulation that results in LTD is accompanied by loss of synaptic connections and that smaller synaptic contacts were most likely to be eliminated. This method will allow us to test our ideas on the molecular mechanisms allowing LTD to 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 diseases such as autism, schizophrenia, and Alzheimers disease.