脳活動は多数のニューロンの発火により生じる．多数のニューロンの時空間的な活動を同時にみるためにはどうすればよいか．その答えの一つが機能性の蛍光色素を用いたイメージングである．われわれは主にカイコガの嗅覚系一次中枢である触角葉(antennal lobe; AL)を対象に，フェロモンを含め，匂いの識別のしくみを探るため，匂い刺激により生じる触角葉の時空間活動をイメージング技術により計測している(図.？)．
神経系の生理機能計測のためのイメージングとしてもっとも普及しているのがカルシウム感受性蛍光色素を用いたものである．これは，ニューロン内にカルシウム感受性色素を負荷して蛍光強度変化を計測するものである．この色素はカルシウムと配位結合することにより，分子の立体構造が大きく変わり蛍光強度が変化する．ニューロンの前/後シナプス活動にはカルシウム濃度変化が伴うため，細胞内にカルシウム感受性色素が存在していればこれを時空間的に観察することができる．色素を注入する方法の一つにAM法がある．AM体（アセトキシメチルエステル体）は膜透過性であり，細胞内のエステラーゼによって細胞内でAM基がとれて，本来の機能を発揮するようになるため細胞外から負荷できる．ただし，細胞内のカルシウム濃度変化とカルシウム蛍光色素の性質により時間スケールは遅く，in vivoでは１秒程度である．われわれはカイコガ触角葉で，匂い刺激に対する応答を計測している（図？）．
最近では，後述のように遺伝子操作技術により特定のニューロンにカルシウム感受性タンパク（たとえばGCaMP）を発現させて計測できるようになり，カイコガのフェロモンリセプタにGCaMPを発現させ，そのイメージングも可能となっている．
一方，膜電位感受性色素は細胞膜表面に結合し，ニューロン活動の興奮・抑制（膜電位変化）による細胞膜の物性変化により，蛍光強度が変化する．その変化幅は小さく，わずかに0.1%程度であるが，膜電位を直接みられるのが強みである．われわれは，触角神経（触角葉への入力）の電気刺激により生じる触角葉の時空間応答の計測に成功している(Hill et al., 2003)（図？）．この応答は神経修飾物質の一つであるセロトニン（5HT）の効果により増大し，カイコガのフェロモン応答の感度調整が中枢で行われることがこの研究から明らかになってきた(Hill et al., 2003)（図？）.
■calcium- and voltage- imaging techniques
We addressed above a method of measuring single neuron activities. Intracellular recording has good temporal and spatial resolution. However, intracellular recording can record only at one point of the neuron and thus recording neural activity at multiple sites in a single neuron is not possible. In addition, in order to record multiple neurons several electrodes and amplifiers are needed and at most a few neurons can be recorded simultaneously by this method. The methods for visualizing neural activities below are alternatives to record multiple neurons and/or multiple sites in a single neuron simultaneously. The temporal resolution is typically on the order of milliseconds to seconds, depending on the specific technique. These methods can be used to visualize activity in dissociated cells in culture, tissue slices , and even intact brains.
Visualizing neural activity depends on specialized fluorescent probes that report changes in calcium concentration, or membrane potential. These probes can be organic dyes that are introduced into a neural system prior to performing an experiment or they can be genetically encoded fluorescent proteins stably expressed in transgenic animals. Dyes tend to exhibit better temporal properties and signal-to-noise characteristics than proteins. However, genetically encoded proteins can be targeted to specific cell types in the brain, allowing for the observation of activity in genetically defined neurons.
Calcium-imaging
Intracellular calcium is central to many physiological processes, including neurotransmitter release, ion channel gating, and second messenger pathways. In neurons, calcium dynamics link electrical activity and biochemical events. Thus, changes in calcium concentration can indirectly indicate changes in electrical activity. Fluorescent calcium indicators are dyes, such as Fluo-4 and Fura-2, and fluorescent proteins, such as aequorin and variants of GFP. Data from calcium imaging experiments typically show changes in fluorescence intensity or the ratio of fluorescence intensity at different wavelengths over time, normalized on the initial level of fluorescence. Calcium indicator dyes can be categorized as ratiometric or nonratiometric dyes. Ratiometric dyes are excited by or emit at slightly different wavelength when they are free of Ca2+ compared to when they are bound to Ca2+. Thus they can report changes in Ca2+ through changes in the ratio of their fluorescence intensity at distinct wavelengths. Ratiometric dyes allow investigators to correct for background changes in fluorescence unrelated to calium dynamics, such as artifacts related to photobleaching, variations in illumination intensity, or differences in dye concentration. However, data acquision and measurements are more complicated than with nonratiometric dyes. Nonratiometric dyes report changes in Ca2+ directly though changes in fluorescence intensity. The common nonratiometric indicators Fluo-4 and Calcium Green-1 exhibit predictable increases in fluorescence intensity with increases in calcium concentration. While the direct relationship between fluorecesnce intensity and calcium concentration is sensitive for detecting changes due to calcium binding, the measurement is prone to detecting changes based on dye concentration and experiment-specific conditions. However, nonratiometric dyes tend to be easier to use.Genetically encoded calcium sensors take advantage of the conformational changes that occur in certain endogenous calcium-binding proteins when they bind to calcium. G-CaMP is an example for a nonratiometric probe reporting through direct changes in fluorescence intensity caused by calcium-sensitive changes in the structure of the fluorophore. We use Calcium Green-1 for measuring neuronal activities in antennal lobe, first olfactory center, of the silkmoth.
Voltage- imaging
Techniques that visualize changes in membrane potential are the closest analogs to electrophysiological recordings, as they report voltage changes in neuronal membranes with high temporal resolution. Voltage-sensitive dye imaging (VSDI) is currently the primary method by which scientists visualize changes in transmembrane voltage spatio-temporally. Voltage-sensitive dyes shift their absorption or emission fluorescence based on the membrane potential, allowing a scientist to gauge the global electrical state of a neuron. Unlike with extracellular electrophysiological techniques, it is possible to detect subthreshold synaptic potentials in addition to spiking activity. These dyes also allow simultaneous activity measurements in large populations. Most dyes exhibit small signal changes; the fractional intensity change is in the range of 10-4 to 10-3. Thus, noise has to be minimized to detect a reliable signal. Furthermore, activity-dependent changes in the intrinsic optical absorption and reflection properties of the brain itself can interfere with voltage-sensitive dye measurements. We succeeded in measuringspatio-temporal activity in the silkmoth antennal lobe in response to electrical stimulation of the antennal nerve (Fig.XX; Hill et al., 2003
). This activity is augmented by application of serotonin (5-HT), thus it reveals that sensitivity of the pheromone response is modulated in the silkmoth central nervous system (Fig. 19; Hill et al., 2003)