TOP特別講演(Special Lecture)
Special Lecture
7月25日(木)14:40~15:40 第1会場(朱鷺メッセ 4F 国際会議室)
Multiplex imaging of neural activity and signaling dynamics
Haruhiko Bito(尾藤 晴彦)
Department of Neurochemistry, Graduate School of Medicine, The University of Tokyo

A central goal of neuroscience is to elucidate how information is encoded and processed at the circuit, neuronal ensemble, single neuron, or subcellular structure resolution in vivo. Ca2+ imaging allows the tracking of population dynamics of neuronal activity at the cell soma levels as well as within neuronal compartments. Recently, an array of genetically encoded calcium indicators (GECIs) were developed with a view to achieving this. However, several design challenges still remained, which together presented a substantial bottleneck to quantitatively understand complex activity dynamics of the brain circuits at the cellular and synaptic levels. To specifically address issues of single action potential (AP) detection speed, two-photon imaging depth and combinatorial spectral compatibility, we here rationally engineered a next-generation of linear and quadricolor GECI suite, XCaMPs. Using green XCaMP-Gf, single AP detection was achieved within 3–10 msec of spike onset, enabling measurements of fast-spike trains in parvalbumin-positive interneurons in the barrel cortex in vivo. A non-invasive, subcortical, imaging using red XCaMP-R uncovered somatosensation-evoked persistent activity in hippocampal CA1 neurons. A combinatorial use of XCaMP-Gf, XCaMP-R and a blue XCaMP-B enabled fiber photometric recording of three distinct (two inhibitory and one excitatory) ensembles during pre-motion activity in freely moving mice. Finally, two-photon co-imaging of yellow XCaMP-Y and XCaMP-R allowed in vivo paired recording of pre- and postsynaptic firing in vivo, revealing spatiotemporal constraints of dendritic inhibition in layer 1 in vivo, between axons of somatostatin (SST)-positive interneurons and apical tufts dendrites of excitatory pyramidal neurons. Thus, XCaMPs represent new multiplexable GECIs, with previously unattained high SNR, linear property, and high frequency spike resolution and offer a critical enhancement of solution space in studies of complex neuronal circuit dynamics. In combination with previous studies on artificial activity-dependent synthetic promoters such as E-SARE, our findings collectively provide a novel toolkit to investigate key molecular, cellular and circuit machineries that are essential for coordinating the formation and maintenance of long-term information processing and regulate cognitive behavior in vivo.
7月27日(土)14:20~5:20 第1会場(朱鷺メッセ 4F 国際会議室)
Keiji Tanaka(田中 啓治)

The prefrontal cortex is thought to be critical for flexible control of behavior in primates, but the mechanisms remain largely unknown. As the prefrontal cortex is composed of multiple areas with different anatomical connections, we expect that the comparison of functional roles among the prefrontal areas would help disentangle the processes of flexible behavioral control.
Flexible application of previously learned behavioral rules is required in goal-directed behavior in complicated environment, and the currently relevant rule is often not directly indicated by sensory cues. The Wisconsin Card Sorting Test (WCST) mimics such a situation. We have trained macaque monkeys with an animal version of WCST. In the task, the monkey selected one of three test stimuli by match with the sample stimulus in color or in shape. The matching rule was constant within a block of trials, but changed between blocks. As there was no cue indicating the currently relevant rule, the monkey had to find the rule based on the reward history in recent previous trials. While bilateral lesion of the principal sulcus region (PS), orbitofrontal region (OFC) or anterior cingulate cortex sulcus region (ACCs) resulted in significant degradation of the overall performance, further analyses of the monkeys' performance showed that the reasons of the degradation were different among the lesion groups. Only the PS lesion impaired maintenance of the currently relevant rule; only the OFC lesion impaired rapid learning of rule value from a single success; and only the ACCs lesion impaired slowing responses under uncertainty. Together with results of single-cell recordings in intact monkeys performing the same task, we conclude that these prefrontal areas contribute to the flexible control of behavior by playing individually specific roles.
Effects of lesioning the frontal pole (area 10), which is assumed to be located at the highest level in frontal cortical hierarchy, were unique. Monkeys with the frontal pole lesion didn't show degradation of the WCST performance. Instead, they were more efficient in conflict adaptation (faster reaction time, which means a better control, after an experience of conflict) and less disturbed by experimentally inserted disturbances. These results suggest that the frontal pole works in disengagement of cognitive resource from the current task to new possibilities, whereas posterior prefrontal areas are essential in execution of the current task.
7月27日(土)15:20~16:20 第1会場(朱鷺メッセ 4F 国際会議室)
Masashi Yanagisawa(柳沢 正史)

Although sleep is a ubiquitous behavior in animal species with well-developed central nervous systems, many aspects in the neurobiology of sleep remain mysterious. Our discovery of orexin, a hypothalamic neuropeptide involved in the maintenance of wakefulness, has triggered an intensive research examining the exact role of the orexinergic and other neural pathways in the regulation of sleep/wakefulness. The orexin receptor antagonist suvorexant, which specifically block the endogenous waking system, has been approved as a new drug to treat insomnia. Also, since the sleep disorder narcolepsy-cataplexy is caused by orexin deficiency, orexin receptor agonists are expected to provide mechanistic therapy for narcolepsy; they will likely be also useful for treating excessive sleepiness due to other etiologies.
Despite the fact that the executive neurocircuitry and neurochemistry for sleep/wake switching has been increasingly revealed in recent years, the mechanism for homeostatic regulation of sleep, as well as the neural substrate for ""sleepiness"" (sleep need), remains unknown. To crack open this black box, we have initiated a large-scale forward genetic screen of sleep/wake phenotype in mice based on true somnographic (EEG/EMG) measurements. We have so far screened >8,000 heterozygous ENU-mutagenized founders and established a number of pedigrees exhibiting heritable and specific sleep/wake abnormalities. By combining linkage analysis and the next-generation whole exome sequencing, we have molecularly identified and verified the causal mutation in several of these pedigrees. Biochemical and neurophysiological analyses of these mutations are underway. Since these dominant mutations cause strong phenotypic traits, we expect that the mutated genes will provide new insights into the elusive pathway regulating sleep/wakefulness. Indeed, through a systematic cross-comparison of the Sleepy mutants and sleep-deprived mice, we have recently found that the cumulative phosphorylation state of a specific set of mostly synaptic proteins may be the molecular substrate of sleep need.

1. Funato et al. Forward-genetics analysis of sleep in randomly mutagenized mice.
Nature 539: 378-383, 2016
2. Wang et al. Quantitative phosphoproteomic analysis of the molecular substrates of
sleep need. Nature, 558: 435-439, 2018
7月28日(日)10:50~11:50 第1会場(朱鷺メッセ 4F 国際会議室)
Masayo Takahashi(髙橋 政代)

Various new therapies were often first tried in ophthalmology field and transplantation of ES/iPS-derived cells was also started from the retina. We transplanted iPS cell-derived retinal pigment epithelium (iPSC-RPE) cell sheet in 2014 to one patient with exudative age-related macular degeneration. Replacing the aged retinal pigment epithelium that cause the disease with the rejuvenated normal tissue of the patient herself will lead to fundamental treatment. Four years after surgery, the transplanted autologous cell sheet did not proliferate, the color of the cell sheet did not change. The adjacent photoreceptor layer and choroidal layer (vascular layer under the retina) were maintained. Tumor has not occurred systemically. 
Although the autologous RPE cell sheet is the best therapeutic material scientifically, the cost of cell preparation is huge and the surgical method is relatively difficult. To make the treatment available for many patients including earlier stage ones, transplantation of HLA-matched iPSC-RPE was done for 5 patients in 2017.
With regard to photoreceptor degenerative diseases, we have studied retinal cell sheet transplantation. Transplantation of 3D retina differentiated from ES/iPS cells to retinal degeneration model mice (Rd 1 mouse) resulted in long time survival and maturation of graft retinal cells. We verified the synapse formation with the secondary neurons of the host retina were confirmed morphologically and electrophysiologically using various model mice. We think that proof of concept (POC) was obtained for photoreceptor transplantation.
Especially there is no method to objectively evaluate the number of synapses. We developed a program that count the mature synapse using imageJ and Naïve Bayes Classifier. With this, we investigated the process of synapses formation in the normal development retina and after transplantation, suggesting that light may promote synapse formation. Now we are preparing for clinical research and clinical trials in cooperation with a company. Photoreceptor transplantation, will be the first example of reconstruction of the neural network of the central nervous system.
On the other hand, prosthesis (artificial retina) is approved and clinical trials of optogenetic therapy is under way in US for retinitis pigmentosa. In addition, the development of devices that compensate for visual impairment is rapid. I will talk about the current status of treatment of retinal degeneration.