Name: In Vivo Imaging of GCaMP6s to Study Rat Trigeminal Ganglion Neurons
Uploaded: 2024-02-09T13:35:00
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in vivo imaging of gcamp6s to study rat trigeminal ganglion neurons

カルシウムインジケーターを用いたラット三叉神経節のin vivo可視化

Somatosensation, the neural encoding of mechanical, thermal, and chemical stimuli impinging on the skin or other bodily structures, including muscles, bone, and viscera, starts with activity in primary afferent neurons that innervate these structures1. Single unit based electrophysiological approaches have provided a wealth of information about the afferent subtypes involved in this process as well as how their stimulus-responses properties may change over time1,2,3. However, while there remains strong evidence in support of the labeled line theory, which suggests specific sensory modalities are conveyed by specific subpopulation(s) of neurons, the ability of many subpopulations of neurons to respond to the same types of mechanical, thermal, and chemical stimuli suggests the majority of somatosensory stimuli are encoded by multiple subpopulations of neurons4,5. Thus, a better understanding of somatosensation will only come with the ability to study the activity of 10&#39;s, if not hundreds, of neurons simultaneously.
Advances in optical approaches with the relatively recent advent of confocal and, subsequently, multiphoton and digital imaging techniques have facilitated the ability to perform relatively non-invasive population-level analyses of neuronal activity6,7. One of the last hurdles in the application of this technology has been the development of tools to enable the optical assessment of neural activity. Given the speed of an action potential that can start and end in less than a millisecond, a voltage-sensitive dye with the capacity to follow changes in membrane potential at the speed of an action potential would be the ideal tool for this purpose. But while there has been tremendous progress in this area7,8,9,10, the signal-to-noise ratio for many of these dyes is still not quite high enough to enable a population analysis of hundreds of neurons at the single cell level. As an alternative approach, investigators have turned to monitoring changes in intracellular Ca2+ concentration ([Ca2+]i). The limitations with this strategy have been clear from the start and include the fact that an increase in [Ca2+]i is an indirect measure of neural activity11; that an increase in [Ca2+]i may occur independently of Ca2+ influx associated with the activation of voltage-gated Ca2+ channels (VGCCs)12,13; that the magnitude and duration of a Ca2+ transient may be controlled by processes independent of VGCC activity11,12,14; and that the time-course of Ca2+ transients far exceeds that of an action potential15. Nevertheless, there are a number of significant advantages associated with the use of Ca2+ as an indirect measure of neural activity. Not the least of these is the signal-to-noise ratio associated with most Ca2+ indicators, reflecting both the magnitude of the change in intracellular Ca2+ and the fact that the signal is arising from the three-dimensional space of the cytosol rather than the two-dimensional space of the cell membrane. Furthermore, with the development of genetically encoded Ca2+ indicators (GECI&#39;s), it is possible to take advantage of genetic strategies to drive the expression of the Ca2+ indicators in specific subpopulations of cells, facilitating population-level analyses in intact preparations (e.g., see16).
Given the number of genetic tools now available in mice, it should be no surprise that GECI&#39;s have been used most extensively in this species. Mouse lines with constitutive GECI expression in subpopulations of sensory neurons have been developed7,16,17. With the development of mouse lines expressing recombinases in specific cell types, it is possible to use even more sophisticated strategies to control GECI expression15. However, while these tools are ever more powerful, there are a number of reasons why other species, such as rats, might be more appropriate for some experimental questions. These include the larger size, facilitating a number of experimental manipulations that are difficult, if not impossible, in the smaller mouse; the ease of training rats in relatively complex behavioral tasks; and at least some evidence that biophysical properties and expression patterns of several ion channels in rat sensory neurons may be more similar to that observed in human sensory neurons than are&#160;the same channels in mouse relative to the human18.
While the transduction of somatosensory stimuli generally occurs in the peripheral terminals of primary afferents, the action potential initiated in the periphery must pass through the structure that houses primary afferent somata, referred to as dorsal root (DRG) or trigeminal (TG) ganglia before reaching the central nervous system19. While there is evidence that not every action potential propagating along a primary afferent axon will invade the cell body20, a consequence of the fact the primary afferent somata are connected to the main afferent axon via a T-junction19, the majority of action potentials initiated in the periphery appear to invade the soma21. This confers three experimental advantages when using GECIs to assess population coding in primary afferents: the large size of the cell body relative to the axons further increases the signal to noise when using [Ca2+]i as an indirect measure of afferent activity; the DRG are generally easy to access; and assessing activity at a site that is spatially remote from the afferent terminals minimizes the potential impact of the surgery needed to expose the ganglia on the stimulus-response properties of the afferent terminals. However, because TG are located beneath the brain (or above the palette), they are far more difficult to access than DRG. Furthermore, while there are many similarities between DRG and TG neurons, there is a growing list of differences as well. This includes the roughly somatotopic organization of neurons in the TG22, unique structures innervated, different central terminal termination patterns23,24,25,26, and now a growing list of differences in both gene expression27,28 and functional receptor expression29. In addition, because we are interested in the identification of peripheral mechanisms of pain, the relatively large number of pain syndromes that appear to be unique to the trigeminal system (e.g., migraine, trigeminal neuralgia, burning mouth syndrome) that appear to involve aberrant activity in primary afferents30,31,32, suggests that the TG needs to be studied directly.
Thus, while stimulus-response properties of TG neurons have been studied with GECIs in the mouse16, because the reasons listed above suggest that the rat may be a more appropriate species to address a variety of experimental questions, the purpose of the present study was to develop an approach to use GECIs to study TG neurons in the rat. To achieve this, we utilized a viral approach to drive the expression of the GECI GCaMP6s in the peripheral nervous system. We then removed the forebrain to allow access to the TG. Finally, mechanical and thermal stimuli were applied to the face while neuronal responses were assessed under fluorescent microscopy. Together, these data support a role for utilizing the rat to investigate changes in the TG under many states, expanding the toolkit for investigators interested in sensory coding in the trigeminal system.

著者スポットライト:三叉神経損傷における神経因性疼痛の末梢メカニズムの探索 

生体内 ラット三叉神経節における感覚ニューロンのカルシウムイメージング

The general goal of our lab is to understand the peripheral mechanisms of pain, and my project specifically investigates pain associated with trigeminal nerve injury. The preparation that I'm showing today allows us to characterize changes in sensory coating and identify afferent subpopulations contributing to neuropathic pain. So we have shown that there are differences in between the trigeminal and somatic afferents in response to nerve injury.NaV1.1 specifically is preferentially upregulated in the trigeminal nerve, suggesting that selective blockers for NaV1.1 can be used to identify different afferent subpopulations that contribute to neuropathic pain. So transgenic mice are used most extensively at the moment in combination with a variety of imaging and omic based strategies. Translating data generated in mice to other species, if not humans.Mice are really small, they're difficult to train, and there's a growing list of differences between mice, rats, and humans. So using rats may help address several of these challenges. Pain mechanisms and potential therapeutic targets differ in craniofacial structures, as in those above the neck, and other somatic and visceral structures, i.e.those below the neck. Results with this preparation suggest that there's a component of neuropathic pain, which is previously thought to reflect changes in the central nervous system, but may actually also reflect changes in the peripheral nervous system.

Watch this Scientific Journal Video about In Vivo Imaging of GCaMP6s to Study Rat Trigeminal Ganglion Neurons at JoVE.com

In Vivo Imaging of GCaMP6s to Study Rat Trigeminal Ganglion Neurons  

ラット三叉神経節ニューロン研究のためのGCaMP6のin vivoイメージング

まず、25マイクロリットルのハミルトンシリンジを使用して、麻酔をかけたラットの仔に15マイクロリットルのAddgeneを腹腔内に注射します。次に、麻酔をかけた生後6〜8週の子犬の毛とひげを剃ります。耳棒付きの脳定位固定装置フレームにラットを取り付けます。そして、動物の体温を維持するために、下に温熱パッドを置きます。次に、出血を最小限に抑えるためにラットの頭に氷冷した生理食塩水に浸したガーゼの場所。サイズ15のメスで、頭蓋骨の上の皮膚と筋肉の正中線を切開します。皮膚と筋肉を引き離して頭蓋骨を露出させます。1×4の丸いドリルビットで、頭蓋骨キャップに慎重に穴を開けて前脳を露出させます。次に、ロンゲールを使用して頭蓋骨を切り裂きます。サイズ15のメスで、脳と嗅球を切開します。へらを使って、硬膜を頭蓋骨から慎重に外します。次に、切断された脳をそっと持ち上げて、三叉神経節と頭蓋底を表示します。出血した出血を焼灼ペンで焼灼します。GCaMP6-Xを画像化するには、頭蓋腔を顕微鏡の対物レンズの下に置きます。次に、末端神経節に焦点を合わせます。次に、顔の1平方センチメートルに機械的刺激を加えます。20倍の対物レンズを使用して、刺激に反応する三叉神経節のニューロンを可視化します。取得ソフトウェアで経時的に蛍光スタトを収集するには、取得メニューの下にあるストリーム取得ボタンを押します。次に、フレーム数を 300 秒、取得時間を 90 秒に設定します。カメラのパラメータを設定して、フレームレートで画像を取得します。3分間にわたって安定したベースライン記録を取得します。ウイルス力価が大きいほど、ラットの仔の神経感染が増加しました。軽い機械的刺激は、三叉神経節のV2領域のみを活性化した。安静時蛍光はほとんどのニューロンで比較的低く、自発的な増加はほとんどありませんでした。自然刺激、特に中断刺激は、最も強力なニューロン応答を誘発しました。慢性狭窄損傷モデルにおけるブラシ刺激は、反対側と比較して、損傷者のピーク反応の大きさが2倍に増加しました。2つの刺激を直列に加えると、2回目の刺激に反応して有意な増幅が見られました。

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Neuroscience

In Vivo Imaging of GCaMP6s to Study Rat Trigeminal Ganglion Neurons

In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their ...