【作者】 郭飞；

【导师】 孙心德； 周晓明；

【作者基本信息】 华东师范大学 ， 生理学， 2012， 博士

【摘要】 自从Hubel和Wiesel在上个世纪60年代研究发现视觉经验影响眼优势柱以来,研究者们在神经元的可塑性上做了很多的研究,并取得了一定的成果。后天经验可以重塑神经元的结构和连接,并且影响到它们的生理和行为；感觉经验和学习可以诱导感觉大脑皮层的可塑性。近年来,感觉经验可塑性的细胞分子机制倍受关注。虽然人们对于经验依赖性的大脑皮层神经元生理特性变化的研究取得了很大的进展,但是对于大脑皮层的片层结构突触的变化和分子机制还知之甚少。本论文通过在体外获取内侧膝状体和听皮层联合脑片,了解下级核团到大脑皮层的神经通路和初级听皮层的片层结构分布。然后通过听觉训练成年大鼠,来探讨行为训练对听皮层的片层结构突触可塑性的影响。最后,探讨听觉强化训练可能的分子机制。论文主要分为以下三个部分：一、内侧膝状体到听皮层的神经通路内侧膝状体上传的听觉信息在听皮层进行加工和整合。解剖学和生理学的研究表明,听皮层的神经纤维投射主要来自于内侧膝状体的腹侧部,膝状体的传入神经到达听皮层第四皮层(layer4),然后通过layer4的神经纤维投射到第三层(layer3)和第二层(layer2)皮层。Layer3和layer2的锥体细胞(pyramidal cells)发出的下行轴突终止于第5(layer5)和第6(layer6)层皮层。Brander等人(Brander,1990)的研究表明：内侧膝状体发出的上行性投射纤维,决定听皮层的音频构筑。尽管有大量的解剖学和生理学的依据,但是来自于内侧膝状体的投射传递到听皮层,并在听皮层里进行加工处理的细胞和突触的机制还知之甚少。在体的胞内记录技术取得了一定的进展,但是由于在电生理和药理学研究的精确性上存在困难,脑片技术的应用可以为胞内和药理学的研究提供了一个理想的离体环境。如果在脑片上,我们可以获得内侧膝状体到听皮层完整的神经纤维连接,就可以为中脑到皮层的内生性、药理学的研究提供方便。在这里,我们以小鼠为实验材料,切取完整的包含内侧膝状体和大脑皮层的联合脑片(thalamocortical brain slice),该脑片包含主要的内侧膝状体的腹侧部核团和初级听皮层,并能保持完整的由内侧膝状体到听皮层的上行纤维。通过电刺激内侧膝状体的腹侧部(ventral division of medial geniculate, MGv)或者内侧膝状体通路,在大脑皮层可以记录到诱发反应。实验结果表明,内侧膝状体的腹侧部和初级听皮层之间的神经纤维联系具有严格的区域定位模式。刺激MGv的某点,可以在听皮层的某一对应区域产生诱发反应,但是最大且潜伏期最短的反应只发生该区域里的某一点,并位于听皮层的layer4。同时,如果刺激MGv不同的部位,在听皮层记录到的最大且潜伏期最短的反应位置也会发生改变,但是最大反应仍然位于layer4。这说明由内侧膝状体发出的神经纤维达到听皮层,具有严格的音频构筑。最后,为了检测该脑片是否仍然具有药理上的功能性。我们把脑片置于尼古丁终浓度为0.2%的人工脑脊液(ACSF)液中,可以观察到听皮层的诱发反应得到明显的加强,并且这种加强产生在整个皮层的激活区域,不管是上皮层(supergranular),中皮层(granular)或者是下皮层(infragranular)。我们的结果显示,该脑片包含有完整的内侧膝状体和听皮层的神经纤维连接,并且这种脑片具有药理学上的功能性。内侧膝状体和听皮层联合脑片对于阐明听觉系统的基本生理机制具有重要的意义,特别是对于听觉系统药理学和各皮层片层间的不同生理功能的研究提供一个良好的体外模型。二、听觉训练增强初级听皮层目标纯音过表达区域皮层间的连接听觉经验训练对听觉可塑性的影响在上个世纪90年代初起开始有大量的报道。Recanzone等对猴子进行几周的纯声训练后,然后让猴子从一系列的声音频率中分辨出该训练频率,结果发现猴子的分辨能力得到明显的提高。对初级听皮层的频率拓扑结构分布分析发现,目标频率在初级听皮层的表达区域增加了,且感受野(receptive field)变窄,潜伏期变短(onset latency)等。Weinberger等对大鼠进行条件刺激(CS+),同样也可以诱导成熟听皮层特殊频率感受野(frequency-specific receptive field, RF)的可塑性,其结果表现在最佳频率(best frequencies, BF)会向CS+的频率偏转,而对于其他频率的声音反应强度减弱了。听觉的可塑性变化在动物出生后的不同时期又有所不同。大鼠生后关键期的被动感觉经验(passive sensory experience)会影响到初级听皮层的频率拓扑结构和电生理的反应特征等,但是关键过后这种被动的经验不会诱导相应可塑性的变化。成年动物的大脑皮层要经历可塑性的变化,听觉经验的刺激必须加上相应的行为训练。在这里,我们用一个5kHz的纯声训练(tone-detection task)成年大鼠,以获得食物的奖赏。动物经历了14天的行为训练,结果显示：训练后的第9—14天,动物对于5kHz的声音识别达到了相当高的水平。对训练后动物的初级听皮层特征频率的拓扑结构分析显示：5kHz的代表区域明显高于对照组,而10kHz的区域明显减少,说明训练后初级听皮层经历了可塑性的变化。之后,我们通过电流源密度的分析(current-source density, CSD)考查听觉训练诱导初级听层可塑行是否影响皮层间的连接?或影响下级核团到皮层的上行投射?或者是二者兼有?通过对各片层结构的CSD感受野(CSD receptive fields, CSD-RFs)的分析,可推知皮层间的多突触连接和内侧膝状体到听皮层的单突触连接的改变。当我们对初级听皮层频率分布的拓扑结构做分析后,16通道的硅制电极被插到～10kH和～20kHz的表达区域,并通过一系列的纯音诱发场电位,然后通过公式换算成CSD。我们分别对这三个片层(layer4, layer2/3, layer6)反应感受野进行分析,结果显示：在～10kHz的记录点,考查layer2/3的CSD-RFs,发现行为训练组的5kHz诱发的电穴得到明显增强(注意到5kHz是目标声音),同时感受野的宽度(阈上10dB)也明显增加。在layer4记录到的内侧膝状体的上级投射对听皮层反应所做的贡献,发现行为训练并未增加5kHz诱发的电穴。同样,对layer6的CSD-RFs分析发现,训练组和对照组基本保持一致。对～20kHz记录点的CSD-RFs分析结果显示：训练组和对照组的layer2/3、layer4和layer6的感受野都保持一致。最后,我们考查动物行为的表现能力与CSD的变化是否有相关性?我们的结果提示,layer2/3的CSD变化跟行为表现能力有很强的相关性(r2=0.763)。综上,我们的研究结果提示,皮层间(intracortical pathway)的多突触连接对于初级听皮层的经验依赖性的可塑性有特殊重要的作用。三、听觉强化训练修复幼年噪声暴露所致空间分辨能力损伤的可能分子机制本实验采用听觉空间的强化训练,探究生后早期噪声暴露造成动物听空间分辨能力的降低,成年后,是否可以通过该行为训练使其恢复,然后通过考察听皮层兴奋性和抑制性受体的表达变化,来探讨其可能的分子机制。动物在关键期被暴露在中等强度的连续噪声下,待动物成年后,通过听空间分辨行为的测试发现,噪声暴露的动物正确分辨空间方位角所用的时间(30-35天)明显高于年龄匹配的正常条件下饲养的动物(10-12天)。尽管如此,通过长时间的强化训练,噪声暴露后的成年动物对于空间方位角的分辨能力仍然可以达到正常动物的水平。为此,我们对其兴奋性受体和抑制性受体的表达水平做了测试,发现噪声暴露明显降低了GABAA受体α1、β2、β3亚单位、以及AMPA受体GluR2亚单位的表达水平,但是GABAA受体α3亚单位表达水平显著提高。重要的是,噪声暴露后的动物,成年后经过听觉空间的强化训练后,这些兴奋性和抑制性受体亚单位的表达水平跟对照组年龄匹配的成年动物的表达水平一致。这个结果表明,早期噪声暴露造成中枢兴奋性和抑制性信息传递系统发育的失衡,成年后可以通过听觉空间的强化训练,调节听皮层兴奋性和抑制性受体亚单位的表达,提高突触传递的效率,修复幼年时不良的听觉环境所造成的听空间分辨能力的损伤。更多还原

【Abstract】 Hubel and wiesel revealed the effect of visual experience on ocular dominance columns in the1960, researchers have been coming into notice that how neuronal architecture and connectivity were shaped by experience in the way that impact their physiology and behavior. Lots of studies have shown that sensory experience and learning could produce the plasticity of sensory brain cortex. Recently, the mechanism underlie experience-dependent plasticity has been widely concerned. However, the laminar profiles of training-induced changes are not well understood, although the research in changes of the physiological properties of neurons induced by experience in the cortex have made great progress. Here, auditory thalamocortical slice was obtained to understand the pathway of the projects from thalamus to cortex and the distribution of neurons in primary auditory cortex. Then we want to know the effect of behavioral training on the laminar profile in trained adult rats. Finally, the mechanism was addressed underlie intensive auditory training.This dissertation including three chapters as follows:Chapter1Auditory thalamocortical pathwayAcoustic information via thalamus is integrated and processed in auditory. Studies in anatomy and physiology have demonstrated that thalamic afferents arrive in layer4(L4), whose neurons project to L2and L3. Axonal projections of pyramidal cells in these layers terminate in L5and some of those from L5in L6. Brander et al. study showed that the forward afferents from thalamus determine the tonotopic organization. Although lots of anatomic and physiological evidences, cellular and synaptic mechanism by which thalamic inputs are transmitted to and processed in ACx are little know. Depite some progress has been made in intracellular recordings in vivo, the difficulty of the precision of electrophysiology and pharmacology still exist. The application of brain-slice preparation provides an ideal environment to do research in intracellular recording and pharmacology. The auditory thalamocortical brain slice maintains an intact auditory thalamocortical pathway enable researcher to facilitate similar progress in the understanding of auditory forebrain mechanisms.Here, mice were use to obtain thalamocortical brain, which contained main ventral of medial geniculate (MGv) and primary auditory cortex and intact forward thalamocortical afferents. Responses were evoked in auditory cortex by MGv stimulation. The result showed that the connection between MGv and primary auditory cortex has strict "zone" model that is the stimulation of MGv could produced responses in a small zone, but the fast and strongest response only in certain point located in middle layer. Also, the position of fast and strongest response would relocate as the position of MGv stimulation was moved, but the response was still in middle layer. This result suggested that projects from thalamus to the middle layer in primary auditory cortex determine the strict tonotopic organization.Finally, nicotine was used to test functional properties of auditory thalamocortical brain slice. Result showed the nicotine enhanced the responses in all layers, which confirmed our hypothesis that this primary thalamocortical slice was functional connection between MGv and primary auditory cortex. The primary slice will provide useful tools to the investigate mechanism of information processing and pharmacological studies in auditory cortex.Chapter2Auditory-cued training strengthens intracortical pathways in primary auditory cortex that mediate response to the rewarded toneThe effect of auditory training on the plasticity in auditory cortex has been widely reported since1990s. Earlier studies in auditory cortex has shown that have shown that training dependent changes in primary auditory map organization after training monkey on a frequency discrimination task. Monkeys significantly improved to discriminate different frequencies after several weeks of behavioral training. Weinberger and colleagues revealed another type of training induced cortical plasticity. Classical conditioning (CS+) induced frequency-specific receptive field (RF) plasticity, which characterized as a shift in the best frequencies in the direction of the frequency of the CS+, and decreased responses to most other frequencies.These studies suggest that auditory training induced plasticity in A1. However, the training-induced plasticity would be different in early and late life. Several studies demonstrated that passive sensory experience influenced the tonotopic organization and electrophysiological properties during critical period. In later life, such plasticity is slower and more limited in adults, except when stimuli are behaviorally relevant. In this study, tone-detection task was used to train rats to get food rewards. The tonotopic organization analysis in primary auditory cortex show that5kHz tone detection training resulted in the5kHz characteristic frequency (CF) representation in A1was expanded while the-10kHz representation significantly reduced relative to naive control rats.In the first chapter, the functional thalamocortical brain slice was obtained and showed main thalamic afferents arrive at layer4, whose neurons project to layer2/3. Here, current-source density (CSD) was used to analyze laminar profiles in A1. Our goal was to determine the laminar profile of changes near the expanded5kHz regions by determining "CSD receptive fields," and infer changes to thalamocortical and/or intracortical inputs.After mapping, we then placed a16-channel silicon multiprobe in middle-(-10kHz) and high-(-20kHz) CF regions and obtained current-source density (CSD) profiles evoked by a range of tone stimuli (CF±1-3octaves in0.25octave steps). Our goal was to determine the laminar profile of changes near the expanded5kHz region by determining "CSD receptive fields," and infer changes to thalamocortical and/or intracortical inputs. Behavioral training altered CSD receptive fields at the10kHz, but not20kHz, site. At the10kHz site, current sinks evoked by a5kHz tone (the target stimulus) were enhanced in layer2/3, but not layer4, and the bandwidth of the layer2/3current sink RF was increased; these results imply training-induced plasticity along intracortical pathways. The layer4current sink receptive field was not clearly separable into thalamocortical and intracortical components, but the results implied lesser, if any, changes to thalamocortical inputs. Finally, we related behavioral performance (d’) to CSD changes in individual animals, and found a strong correlation between d’on the final day of training and the amplitude of the5kHz-evoked current sink in layer2/3(r2=0.763).The prediction of behavioral performance by the target-evoked layer2/3current sink suggests that this intracortical pathway is important for brain plasticity underlying learning.Chapter3Intensive training in adults refines auditory discrimination degraded in early life and alters expression of GABAA subunits and GluR2in auditory cortexPerceptual training has important role in the functionally development in the auditory cortex. In this chapter, rats were trained to examine whether discrimination degraded in early life could be recovered by intensive sound-azimuth spatial training. Then western blotting were used to investigate mechanism underlie learning.Rat pups were raised in the presence of continuous and moderate level pulsed noise during critical period. At postnatal35, go/no-go the sound-azimuth spatial strategy was used to train rats and control rats for approximate4weeks. Behavioral results have shown that the time course for noise-reared rats to master the behavior was30-35days, which was remarkable more than control group that was10-12days. The results indicated the that intensive spatial target training significantly improved behavioral performing for noise-reared animals even though it took a longer time to catch up the same level as the control animals. Further more, western blotting was used to examine the changes of the inhibitory and excitatory receptor. Then results showed that the pulse-noise exposure resulted in decreasing the expression of the GABAA receptor a1subunit, AMPA receptor GluR2subunit and GABAA receptor β2and GABAA receptor (33subunit. However, the expression of GABAA receptor a3increased. More important, we found that intensive spatial training in adulthood would result in a return expression level as the control adult animals. The results further demonstrate the perceptual training, as a strategy functional would normalize the deteriorated auditory cortex in adults in the basis of molecular mechanisms.更多还原