Symposium
Voltage imaging: What's New?
シンポジウム
膜電位イメージング:新展開 |
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| 7月26日（金）15:10～15:34 第6会場（朱鷺メッセ 2F 201A）
2S06a-1
Simultaneous dendritic voltage and calcium imaging and somatic recording from Purkinje neurons in awake mice
Bernd Kuhn（Kuhn Bernd），Christopher J. Roome（Roome Christopher J.）
Okinawa Institute of Science and Technology Graduate University
Spatiotemporal maps of dendritic signalling and their relationship with somatic output is fundamental to neuronal information processing, yet remain unexplored in awake animals. Here, we combine simultaneous sub-millisecond voltage and calcium two-photon imaging from distal spiny dendrites, with somatic electrical recording from spontaneously active cerebellar Purkinje neurons (PN) in awake mice. We use the purely electrochromic, synthetic voltage-sensitive dye ANNINE-6plus. A chronic cranial window with access port allows us to load the PNs by electroporation. Due to the spectral properties of ANNINE-6plus, it is possible to simultaneously image calcium with the genetically encoded indicator GCaMP6f. This technique allows simultaneous voltage and calcium imaging from dendrites for many minutes at 2kHz temporal resolution. The labeling lasts for up to two weeks with a single electroporation. We detect discrete 1 to 2 ms suprathreshold voltage spikelets in the distal spiny dendrites during dendritic complex spikes. Spikelets and their calcium correlates are highly heterogeneous in number, timing and spatial distribution within and between complex spikes. Back-propagating simple spikes are highly attenuated. Highly variable 5 to 10 ms voltage hotspots are localized to fine dendritic processes and are reduced in size and frequency by lidocaine and CNQX. Hotspots correlated with somatic output but also, at high frequency, trigger purely dendritic calcium spikes. Summarizing, spatiotemporal signalling in PNs is far more complex, dynamic, and fine scaled than anticipated, even in resting animals. | | 7月26日（金）15:34～15:58 第6会場（朱鷺メッセ 2F 201A）
2S06a-2
膜電位イメージングにより捉えるゼブラフィッシュ小脳の発達
Sachiko Tsuda（津田 佐知子）
埼玉大院理工
The general anatomical organization of the cerebellar neural circuits is well defined; however, their functional organization and its development are still unresolved. Recent advances in optical techniques provide powerful tools to address this issue. Among them is voltage imaging which enables a direct measurement of neural activity from a population of neurons constituting neural circuits.
Here, we have applied genetically encoded voltage indicators (GEVIs), ASAP1 (Accelerated Sensor of Action Potentials 1) and QuasAr2 (Quality superior to Arch 2), to zebrafish, which is known to have cerebellar circuitry similar to that of mammals, and whose transparency and small size provide a big advantage in applying optical techniques. First, we established transgenic lines, UAS:ASAP1 and UAS:QuasAr2-mOrange, which express the voltage sensors by Gal4-UAS system. By using several gal4 lines, we concluded that ASAP1 was properly expressed in zebrafish neurons including cerebellar neurons. However, QuasAr2 signal could not be detected. Next, to examine whether neuronal activity could be detected by ASAP1, we performed electrical stimulation of the cerebellum while resulting neuronal activity was recorded by high-speed and whole-cerebellar imaging. With a transgenic fish that expresses ASAP1 in neurons, we detected the evoked depolarization widely in the cerebellum and tectum, which was also confirmed by electrophysiological recordings. Furthermore, we succeeded in recording the spontaneous activity of spinal cord neurons at single-cell resolution. These responses were significantly reduced by treatment with tetrodotoxin, leading to the conclusion that ASAP1 enables an optical measurement of neuronal activity in zebrafish brain including the cerebellum. We also established voltage sensitive dye imaging in the cerebellum of zebrafish larvae using Di-4-ANEPPS, and showed that it could detect both depolarization and hyperpolarization in the developing cerebellum. This approach will enable a detailed analysis of the spatiotemporal dynamics of the excitation and inhibition in the cerebellum. Combination of these imaging with other approaches such as optogenetics and behavioral analysis would help deeper understanding of the functional organization of the cerebellar circuitries and its development. | | 7月26日（金）15:58～16:22 第6会場（朱鷺メッセ 2F 201A）
2S06a-3
遺伝子にコードされた膜電位センサーとその応用
Masayuki Sakamoto（坂本 雅行）
東京大院医神経生化学
Imaging membrane potential in neurons has become a fruitful approach to study neural circuits. Recently, the effective performance of genetically encoded fluorescent voltage indicators has been greatly improved. Here, we developed a novel genetically encoded voltage indicator, ArcLight-MT, and demonstrated the use of it. First, we applied it to measure the voltage in dendritic spines. Dendritic spines have a key role for the function of cortical circuits since they mediate most excitatory inputs. While it is demonstrated that they serve to compartmentalize calcium, it is still unclear if they have an electrical function, compartmentalizing voltage and electrically modifying the excitatory post synaptic potentials (EPSPs). Although this potential role as an electrical compartment could have wide implications for our understanding of synaptic transmission, there are unfortunately scant data whether spines can directly modify membrane potential. Due to the small size of spines, it is not possible to measure their electrical properties with standard electrophysiological techniques. To examine electrical properties of dendritic spines, we recorded voltage fluorescence signal in response to synaptic input with two-photon glutamate uncaging in mouse hippocampal cultured neurons. We found that EPSPs generated by glutamate uncaging were attenuated by up to 4-fold as they propagate to the parent dendrites. Our results suggest that spine heads are electrically isolated by the spine neck from membrane potential signals in parent dendrites. The attenuation of EPSPs by spines could have important repercussions for synaptic plasticity and dendritic integration. Next, we utilized this indicator in vivo with two-photon microscopy. We succeeded to record visual stimulus-induced fluorescent response in visual cortex with single cell resolution in single trials. Our results demonstrate that the feasibility of voltage imaging as a tool to monitor the activity of neurons. | | 7月26日（金）16:22～16:46 第6会場（朱鷺メッセ 2F 201A）
2S06a-4
閾値下膜電位変動の検出に特化した蛍光膜電位センサーの開発とその応用
Yuki Bando（阪東 勇輝）
浜松医大医器官組織解剖学
Measuring both synaptic inputs and action potentials from multiple neurons is essential to understand neural computation, but it has been challenging using conventional physiological techniques. To achieve this, voltage imaging is a promising technology. However, unfortunately, current fluorescent voltage indicator cannot detect either sbuthreshold or spiking activity reliably with cellular resolution in vivo because of poor performance of voltage indicators and limitation of imaging speed using two-photon microscope. Here, we developed a genetically-encoded voltage indicator which was specialized for detection of subthreshold membrane potential dynamics. We found that this genetically-encoded voltage indicator could detect subthreshold membrane potential change, but could not detect action potentials with simultaneous two-photon voltage imaging and whole-cell patch-clamp recording in vivo. We then combined two-photon voltage imaging and calcium imaging in vivo, and this simultaneous two-photon voltage and calcium imaging successfully recorded subthreshold inputs and action potentials. We will also discuss application of this technique to a pathophysiological model. Simultaneous voltage and calcium imaging using genetically-encoded indicators could be useful to large-scale analysis of neural circuit function. | | 7月26日（金）16:46～17:10 第6会場（朱鷺メッセ 2F 201A）
2S06a-5
神経回路ダイナミクスのリアルタイム光学計測：膜電位感受性色素（VSD)と早い内因性信号（FIOS)
Takashi Tominaga（冨永 貴志），Yoko Tominaga（冨永 洋子）
徳島文理大神経研
The brain is a vast and precise information processing device composed of a large number of neurons. The information of the neural circuit is encoded in the membrane potential of the cell. In order to understand and interpret this information, the readout of the membrane potential from the elements of the neural circuitry is crucial. Voltage-sensitive dye (VSD) was developed to acquire fast membrane potential changes in the excitable membrane using optical measurement devices as early as the 1970s. Soon, developments of large-scale imaging device enable us to visualize the activity of the neural circuit. However, the simple wide-field VSD imaging methods have long been limited technique for some limited laboratories until the late 1990s, mostly because of its poor signal noise ratio (SNR). In this talk, I will introduce how we solved the technical difficulties, and the VSDI became to be a "conventional" method. The established stability of VSD imaging enables us to quantify the long-term circuit changes such as the long-term potentiation in area CA1 of mouse hippocampal slice over 12 hours. Also, this can apply to the evaluation of far broad areas of neural circuit activities. The talk will introduce the plastic change of the neural circuit activity in the entorhinal and perirhinal cortices. Meanwhile, when looking back at the origin of the optical recordings, we found a new class of intrinsic optical signal associated with neural activation. The most interesting characteristics of the intrinsic signal are the time constant of the response, which is almost comparable to the signal observed with VSD. Pharmacological evidence and physiological characteristics showed that the fast intrinsic optical signal (FIOS) is indeed dependent on the postsynaptic neural circuit activity. Overall, the talk will introduce the use of established VSD imaging and the application of this method. | | | |
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