以微正子斷層造影不同型式刺激在大鼠視丘與大腦皮質之反應與神經信號記錄用多通道微電極之研發

MicroPET Imaging of Thalamic and Cortical Activation in Rat Brain Responding to Different Modalities of Stimulation and Development of Multichannel Electrodes for Neuronal Signals Recording

陳右穎(You-Yin Chen)

指導教授 ： 郭德盛

共同指導教授 ： 趙福杉

國立臺灣大學/電機資訊學院/電機工程學研究所/博士(2004年)

https://doi.org/10.6342/NTU.2004.00576

摘要

神經科學是一門複雜且奧妙的學問，尤其神經中樞-大腦更是眾多學者致力研究的目標，如何運用適當的儀器及技術剖析大腦精細的運作弁鄏足健巨蒗D戰性且富含研究價值的工作。大腦是由上億個神經元細胞所組成的精密器官，它不斷的處理、分析、及認知由外界輸入的訊息，並必須適時反應，才能控制及協調個體的表現。而研究弁鄔吨j腦映射有助於區辨經由感覺 (Sensory)、運動 (Motor)、認知 (Cognitive function) 或是情緒反應 (Emotional process) 所引發之神經活動的特定區域。
本研究之目的在於探討大腦接受外界刺激或行為改變所產生之反應在空間與時間的變化。利用弁鄔宎v像與電生理技術則可提供大腦解剖性與弁鄔吨妞袺鷏穈T。弁鄔宎v像技術以毫米等級 (Millimeter) 的空間精確度定位大範圍神經活動的區域，並以次秒 (Subsecond) 等級的時間解析度確認反應時間。電生理技術則運用更精細的空間(Micrometer)與時間 (Millisecond) 解析度快速且確實記錄神經活動信號。但其缺點在於無法進行大範圍記錄。
本研究使用大鼠尾部作為感覺系統的簡單模型，其原因有兩點。首先，大鼠尾部在腦部的反應區較小，有利於節省人力作深入的研究；再者，大鼠尾部外觀簡單且長，容易外掛刺激器提供各種型式的自然刺激。由於大鼠尾部在腦部所分佈的反應區很小 (1 mm3)，因此大鼠尾部受刺激時需使用具高影像解析度與靈敏度的正子斷層掃瞄評估腦部代謝活動 (Metabolic function) 的改變並獲得其弁鄔宎v像。此外將核醫藥物氟-18去氧葡萄糖 (18FDG) 注射於動物體內以標定大腦反應區，利用微正子斷層造影 (microPET scanner) 提供高解析度的影像分析神經活動所造成組織代謝的改變。本研究使用微正子斷層造影 (microPET) 來分析電刺激 (Electrical stimuli)、機械性刺激 (Mechanical stimuli)、溫度刺激 (Temperature stimuli) 與傷害性刺激 (Noxious stimuli) 在大鼠視丘與大腦皮質的反應。首先，先評估麻醉藥對腦部影像的影響，研究發現使用巴比妥鈉 (Pentobarbital sodium)麻醉動物之影像較使用卡門 (Ketamine) 麻醉動物之影像為模糊。因此為了確保影像判讀正確，選擇卡門作為動物實驗麻醉藥劑。本研究也提供可靠且非侵入式的的腦部定位方法，透過正子斷層造影設備的雷射定位系統對準大鼠頭骨定位點前囪門 (Bregma) 達到精確定位的弁遄C為了造影資料的正規化，使用活化指標 (Activation Index) 做為神經活動造成組織代謝之評估。本研究發現電刺激大鼠單側尾部造成對側大腦皮質與視丘的活化指標比同側腦組織有明顯的差異。機械性刺激單側尾部造成對側大腦皮質的活化指標較同側腦組織有明顯的差異，但比較兩側視丘的活化指標並無明顯差異。其原因可能是微正子斷層設備空間解析度不足而無法比較兩側視丘神經組織代謝，或實驗動物數目不夠無法達到統計上顯著性差異。不同溫度刺激大鼠單側尾部發現刺激溫度為15℃、35℃和40℃皆會造成對側大腦皮質與視丘之活化指標高於同側腦組織。以25℃為控制溫度時，比較其大腦兩側所得之活化指標並沒有顯著性差異。過熱 (> 45℃) 和過冷 (< 6℃) 造成大鼠單側尾部傷害性刺激，發現兩側大腦皮質與視丘神經組織對氟-18去氧葡萄糖的攝取皆提升，但兩側腦組織的氟-18去氧葡萄糖攝取並沒有統計上明顯差異。
此外，使用電生理技術彌補微正子斷層造影在時間解析度的不足。為了記錄更多的神經元活動，本研究依場電位記錄準則(Field potentials)製作多通道的探針電極來記錄視丘場電位信號。本研究所確認的記錄準則皆符合統計意義，在信號平均次數50次以上其交互相關係數 (Coefficient of cross correlation) 可達95 %以上；電極記錄間距沿著AP、ML與VD方向在50 μm內時其交互相關係數皆達95 %以上。由於大鼠尾部在視丘反應區呈現垂直分佈，故利用半導體製程技術與雷射微加工技術製作多通道探針電極 (Multielectrode probe)。其十六個電極設計為垂直排列且兩兩間距為50 μm。此電極探針具有多點偵測信號弁遄A能同時提供多個記錄點的場電位與多神經元活動 (Multiunit activity) 信號。
目前，多通道電極技術已趨成熟，且專為動物造影的正子斷層設備已具有高空間解析度的弁遄C結合影像與電生理技術，將為日後大腦弁鄔坌膍s帶來新契機。尤其在精神心理層面的研究，將藉由精密儀器及創新技術的輔助突破傳統神精科學研究的界線，有助於解開更高層次的大腦皮質弁鄐岐慼C

關鍵字

交互相關係數；多通道微電極探針 ； 場電位 ； 正子斷層 ； 氟-18去氧 葡萄糖 ； 微正子斷層照影 ； 雷射微加工技術 ； 微機電製程

並列摘要

Adequately understanding the brain function is one of the outstanding challenges in neuroscience. The brain is an unresting assembly of cells that continually process spatial distributed information, analyzes it, perceives it, and makes decision. Therefore, the goal of functional brain mapping is to isolate local neuronal activity associated with sensory, motor, and cognitive function or with emotional process.
This study aims to clarify the process mechanism of the brain in the spatial and temporal domains under a given task or behavior. Functional imaging and electrophysiology techniques are powerful tools to investigate the anatomical and functional information on neuronal activity within the brain. A functional imaging needs both millimeter precision in localizing regions of activated tissue and subsecond temporal precision for characterizing changes in patterns of activation over time. Electrophysiological methods can provide the temporal resolution as fine as the analog-to-digital sampling rate (typically in the 1 – to 10-msec range), and its exquisite sensitivity to changes neuronal activity has been recognized.
This study used the rat tail as a simple model of the sensory system. It is chosen for two simple reasons: first, the previous work has found that the receptive field of rat tail is very small (1 mm3), therefore, it is good to save manpower to study it in detail; second, the tail is sufficiently long to easily attach an electrical stimulator or other natural modalities of stimulation. To acquire the functional image of the rat brain, positron emission tomography (PET) with high resolution and sensitivity was adopted to assess the metabolic activity. Using [18F]fluorodeoxyglucose (FDG) as the radiotracer, we demonstrate the high-resolution PET scanner (microPET) has sufficient resolution to image metabolic function of the rat brain as well as to determine patterns of neuronal activation produced by different modalities of stimulation. In this study microPET was used to investigate neuronal activation of thalamic and cerebral cortical responses to electrical stimuli (ES), mechanical stimuli (MS), different intensities of temperature stimuli and noxious stimuli (cold and heat) of the left side of the rat tail. We first evaluated sodium pentobarbital and ketamine to determine their effect on microPET images. Pentobarbital anesthesia significantly reduced FDG uptake in neural tissues, blurring images; therefore, ketamine was use to anesthetize animals during microPET. After rats were anesthetized and secured in a laboratory-made stereotaxic frame, a simple, noninvasive stereotaxic technique was used to position their heads in the microPET scanner and to precisely confirm the images in the stereotaxic atlas. An activation index (AI) represented changes in metabolic activity in neural tissues. ES resulted in more increases in FDG uptake in the contralateral thalamus (AI = 18) and cortex (AI = 12.5), with significant side-to-side differences (P < .05, paired t-test). MS induced more uptakes in contralateral cortex (AI = 9.5), with the significant side-to-side differences (P < .05, paired t-test). However, lateralized differences were absent in the thalamus (P > .05, paired t-test) due to the limited spatial resolution of microPET. In the warm-discrimination study, two intensities of innocuous heat (35℃ and 40℃) were applied to the left side of the rat tail for 30 minutes. Significant increases in FDG uptakes to both 35℃ and 40℃ stimuli were found in the contralateral thalamus and cortex. The results FDG uptakes in contralateral thalamus were also showed the significant discrimination between 40℃ (AI = 17.34) stimulus and 35℃ stimuli (AI = 12.56; P < .05, paired t-test). The innocuous cold (15℃) showed significant side-to-side differences (P < .05, paired t-test) between bilateral thalamus and cortex. The results showed two forms of noxious stimuli (> 45℃ and < 6℃) increase FDG uptakes in bilateral thalamus and cortex but no significant bilateral difference occurs.
To compensate for the insufficiency in the temporal resolution of microPET, this study used the electrophysiological technology to record the firing patterns of neurons one at a time while stimuli were presented. In this study, to record from many neurons simultaneously, a multichannel electrode has been fabricated to record the thalamic field potentials (FPs) responding to the electrical stimulation of nerve at the rat tail. At first, the number of sweeps used to form the evoked FP average and the spatial sampling density were determined by using cross-correlation functions, which were then statistically analyzed. The difference was significant at P < 0.05, if the number of sweeps for averaging was more than 50 and the spatial interval between two consecutive recording sites was less than 50 μm in the anteroposterior, mediolateral and ventrodorsal directions. The responsive area was distributed vertically in the thalamus (ventral posterior lateral [VPL] nucleus); therefore, the recording sites were arranged in one linear array. Sixteen recording sites, which were 50 μm apart from each other, were distributed in the ventrodorsal direction. A 16-channel silicon probe was fabricated by using a standard photolithography process and laser micromachining techniques. The probe provides capabilities to record multiple field potentials and multiunit activities simultaneously.
The multichannel electrode has been developed and high-resolution PET scanner is now available. Combination of imaging with electrophysiological techniques will enhance understanding of mental activities directly, follow their progress in the living brain, and make inferences regarding higher cortical functions. The area will be the wave of the future.

並列關鍵字

Field potential ； Micro-electro-machining ； Laser micromachining ； Positron emission tomography ； FDG ； MicroPET ； Multielectrode probe ； Cross-correlation

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