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

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CAdequately 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.Chapter 1 Introduction 1 1.1. Preliminary 1 1.2. Alternative medical imaging techniques 2 1.3. How is PET different? 4 1.4. PET imaging for brain function 6 1.5. Why image small laboratory animal with PET? 7 1.6. Electrophysiology 8 1.7. Ensemble recording 9 1.8. Dissertation organization 11 Chapter 2 Imaging Brain Function with MicroPET under Different Modalities of Stimulation 23 2.1. Research motivation 23 2.2. Experimental protocol 23 2.2.1. Animal subjects 23 2.2.2. PET Scanner and cyclotron facility resources 24 2.2.3. Evaluation of anesthetics for microPET imaging 24 2.2.4. Stereotaxic technique for imaging the rat brain 26 2.2.5. Functional images for stimulation of the rat tail 27 2.2.5.1. ES and MS of the rat tail 27 2.2.5.2. Stimuli of warm, cold, and nociception 28 2.2.6. Data analysis 29 2.3. Results 31 2.3.1. Effects of anesthetics on microPET images 31 2.3.2. Alignment of microPET images to the stereotaxic atlas of the rat brain 31 2.3.3. Comparison of ES and MS with the non-stimulus control condition 32 2.3.3.1. MicroPET images 32 2.3.3.2. Dynamic FDG uptake in the thalamus and cerebral cortex 33 2.3.3.3. Quantitative analysis 34 2.3.4 Comparison among neuronal activation under the stimuli of warmth, cold, and nociception 35 2.3.4.1. MicroPET images 35 (a) Warm-warm (35℃ vs. 40℃) discrimination 36 (b) Warm and noxious heat (40℃ vs. 45℃) discrimination 36 (c) Cold and noxious cold (15℃ vs. 5℃) discrimination 37 2.4. Discussion 37 2.4.1. Interactions of anesthetics 37 2.4.2. Comparison with stereotaxic technique during microPET imaging 38 2.4.3. Determination of the FDG uptake period during microPET 39 2.4.4. Differences in microPET images with different stimuli 39 2.4.4.1. Comparison ES with MS 39 2.4.4.2. Comparison among stimuli of warmth, cold and nociception 41 Chapter 3 Electrophysiological Methods for Mapping Brain Function: Development of Multichannel Electrodes 63 3.1. Extracellular recording from single cells to ensembles 63 3.2. Considerations for multichannel electrode Design 63 3.3. Materials and methods 65 3.3.1. Animal preparation 65 3.3.2. Stimuli and recording 65 3.3.3. Determination of the neural recording criteria 66 3.3.4. Estimations of SNR 68 3.3.5. Fabricating the multielectrode probe 69 3.4. Results 71 3.4.1. Number of evoked FPs to be averaged and spatial Interval 71 3.4.2. Electrical characteristics of the laboratory-designed probe 72 3.4.3. Comparison with the performance of various electrodes in vitro and in vivo testing 72 3.4.4. Neural recording 73 3.5. Discussion 75 3.5.1. Probe specification based on optimal criteria of recording thalamic evoked FPs 75 3.5.2. Techniques for shaping probe 75 3.5.3. Capability of the laboratory-designed probe 77 Chapter 4 Conclusions and Future Works 88 4.1. Small animal imaging with multiple modalities 88 4.2. Pharmacology of anesthesia 88 4.3. Future of small animal PET technology 89 4.4. Drawbacks of the laboratory-designed probe 89 4.5. More designs and applications of multichannel electrodes 90 4.6. Combination of functional imaging and electrophysiological methods 90 Bibliography………………………………………………………96 Table 1-1. Summary of the different types of experimental preparations that use multiple single-unit electrodes and the different types of electrodes. 21 Table 2-1. MicroPET scanner specifications 44 Table 2-2. Regions were drawn on an image of coronal section (No.34) in the axial direction and ROI sizes and shapes were uniform across the scans. 51 Table 2-3. The statistical results of ROI in thalamus and cerebral cortex. ROIs were drawn accurately by each investigator. The units of mean value is nCi/cc 53 Table 2-4. Comparison of FDG uptake periods in the thalamus and cerebral cortex under non-stimulus control, ES, and MS conditions 56 Table 2-5. Regional differences in FDG uptake expressed as the mean percentage difference from the mean whole-brain activity 57 Table 2-6. Regional differences in FDG uptake expressed as the mean percentage difference from the mean whole-brain activity 60 Table 3-1. Specifications of the multielectrode probe 80 Figure 1-1. A immunolabelled brain of Tritonia diomedea (a sea slug).. 13 Figure 1-2. How the PET scan works. 14 Figure 1-3. 2-fluro-2-deoxy-D-glucose. This positron emitter has a half-life of 109.8 minutes and is the most commonly used radioactive tracer in PET scans. 15 Figure 1-4. Glucose and deoxyglucose in brain tissue 16 Figure 1-5. 18F-FDG rat images obtained with MicroPET, the animal PET developed at UCLA. 18 Figure 1-6. Glass micropipettes 19 Figure 1-7. Compare similarity across two signals recorded in thalamus and cortex at some electrode in different time 20 Figure 1-8. Examples of microfabricated electrode arrays. 22 Figure 2-1. MicroPET is a dedicated PET scanner designed for high resolution imaging of small laboratory animals.. 43 Figure 2-2. Rats in the stereotactic frame are maintained on anesthetics for the duration of microPET studies. 45 Figure 2-3. Coronal FDG microPET images show glucose metabolism.. 46 Figure 2-4. FDG uptake values of various brain tissues under different anesthetic agents 47 Figure 2-5. An anesthetized rat in the stereotactic frame is attached to the microPET bed, and its head is aligned with the microPET laser crosshair. 48 Figure 2-6. MicroPET images for coronal sections through the rat brain are arranged rostral to caudal. 49 Figure 2-7. A rat was positioned within the scanner and scanned for 15 minutes. After removal of the animal from the stereotactic frame, it was repositioned in the same manner and scanned for an additional 15 minutes. 50 Figure 2-8. MicroPET scans during non-stimulus, ES, and MS conditions. 52 Figure 2-9. Time-radioactivity curves show FDG uptake in the thalamus and cerebral cortex under non-stimulus control, ES, and MS conditions. 54 Figure 2-10. Bar graphs show metabolic activity in contralateral and ipsilateral thalamus and cortex under control, ES, and MS conditions. 58 Figure 2-11. MicroPET scans during non-stimulus (25℃), innocuous warm and heat contact stimuli (35℃ and 40℃), noxious heat contact stimulation (45℃), one innocuous cold contact stimulation (15℃), and noxious cold stimulation (5℃). 59 Figure 2-12. This bar compares the side-to-side differences in thalamus under various intensities of temperature. 61 Figure 2-13. This bar compares the side-to-side differences in cortex under various intensities of temperature.. 62 Figure 3-1. Effects of number averaging of evoked FPs and the interelectrode distance between two consecutive recording sites on . 78 Figure 3-2. Schematic view of the multielectrode probe structure. 79 Figure 3-3. Diagram illustrates fabrication of the multielectrode probe. 81 Figure 3-4. Detailed view of the multielectrode probe. 82 Figure 3-5. Impedance spectroscopy of the multielectrode probe 83 Figure 3-6. SNR of evoked FPs recorded from four types of electrodes plotted against various sweeps to average evoked FPs 84 Figure 3-7. Representative examples show that MUAs and evoked FPs 85 Figure 3-8. A exapmle of the laboratory-designed for recording the thalamic FPs and CSDs analysis 86 Figure 3-9. Microcracks distributed on the outermost aspect of the shaft. 87 Figure 4-1. Coronal images of T2-weighted MRI and microPET images. The PET images have been registered to the MRI and are presented with their superimposed images. 92 Figure 4-2. A trace retained on the brain slice after remove the neural probe.. 93 Figure 4-3. Silicon-based forked shape microelectrode arrays for cortical recording and stimulation.. 94 Figure 4-4. New developments will extend the range of applications of currently available passive MEAs.. 9