Tadashi Tsubota

Activation of TGF-beta/activin signalling resets the circadian clock through rapid induction of Dec1 transcripts.

The circadian clock is reset by external time cues for synchronization to environmental changes. In mammals, the light-input signalling pathway mediated by Per gene induction has been extensively studied. On the other hand, little is known about resetting mechanisms that are independent of Per induction. Here we show that activation of activin receptor-like kinase (ALK), triggered by TGF-β, activin or alkali signals, evoked resetting of the cellular clock independently of Per induction. The resetting was mediated by an immediate-early induction of Dec1, a gene whose physiological role in the function of the circadian clock has been unclear. Acute Dec1 induction was a prerequisite for ALK-mediated resetting and upregulation was dependent on SMAD3, which was phosphorylated for activation in response to the resetting stimuli. Intraperitoneal injection of TGF-β into wild-type or Dec1-deficient mice demonstrated that Dec1 has an essential role in phase-shift of clock gene expression in the kidney and adrenal gland. These results indicate that ALK–SMAD3–Dec1 signalling provides an input pathway in the mammalian molecular clock.

Characterization of the Properties of Seven Promoters in the Motor Cortex of Rats and Monkeys After Lentiviral Vector-Mediated Gene Transfer

Lentiviral vectors deliver transgenes efficiently to a wide range of neuronal cell types in the mammalian central nervous system. To drive gene expression, internal promoters are essential; however, the in vivo properties of promoters, such as their cell type specificity and gene expression activity, are not well known, especially in the nonhuman primate brain. Here, the properties of five ubiquitous promoters (murine stem cell virus [MSCV], cytomegalovirus [CMV], CMV early enhancer/chicken β-actin [CAG], human elongation factor-1α [EF-1α], and Rous sarcoma virus [RSV]) and two cell type-specific promoters (rat synapsin I and mouse α-calcium/calmodulin-dependent protein kinase II [CaMKIIα]) in rat and monkey motor cortices in vivo were characterized. Vesicular stomatitis virus G (VSV-G)-pseudotyped lentiviral vectors expressing enhanced green fluorescent protein (EGFP) under the control of the various promoters were prepared and injected into rat and monkey motor cortices. Immunohistochemical analysis revealed that all of the VSV-G-pseudotyped lentiviral vectors had strong endogenous neuronal tropisms in rat and monkey brains. Among the seven promoters, the CMV promoter showed modest expression in glial cells (9.4%) of the rat brain, whereas the five ubiquitous promoters (MSCV, CMV, CAG, EF-1α, and RSV) showed expression in glial cells (7.0-14.7%) in the monkey brain. Cell type-specific synapsin I and CaMKIIα promoters showed excitatory neuron-specific expression in the monkey brain (synapsin I, 99.7%; CaMKIIα, 100.0%), but their specificities for excitatory neurons were significantly lower in the rat brain (synapsin I, 94.6%; CaMKIIα, 93.7%). These findings could be useful in basic and clinical neuroscience research for the design of vectors that efficiently deliver and express transgenes into rat and monkey brains.

A bicistronic lentiviral vector-based method for differential transsynaptic tracing of neural circuits

We developed a bicistronic HIV1-derived lentiviral vector system co-expressing green fluorescent protein (AcGFP1) and wheat germ agglutinin (WGA) mediated by picornaviral 2A peptide. This system was first applied to the analysis of the rat cerebellar efferent pathways. When the lentiviral vector was injected into a specific lobule, the local Purkinje cell population (first-order neurons) was efficiently infected and co-expressed both AcGFP1 and WGA protein. In the second-order neurons in the cerebellar and vestibular nuclei, WGA but not AcGFP1 protein was differentially detected, demonstrating that the presence of AcGFP1 protein enables discrimination of first-order neurons from second-order neurons. Furthermore, WGA protein was detected in the contralateral ventrolateral thalamic nucleus (third-order nucleus). This system also successfully labeled rat cortical pathways from the primary somatosensory cortex and monkey cerebellar efferent pathways. Thus, this bicistronic lentiviral vector system is a useful tool for differential transsynaptic tracing of neural pathways originating from local brain regions.

Optogenetic inhibition of Purkinje cell activity reveals cerebellar control of blood pressure during postural alterations in anesthetized rats

The cerebellar uvula (lobule IX), a part of the vestibulocerebellum, is extensively connected to the areas of the brainstem that participate in cardiovascular regulation and vestibular signal processing. This suggests that the uvula regulates blood pressure (BP) during postural alterations. Previous studies showed that lesions of the uvula affected the baroreceptor reflex and cardiovascular responses during postural alterations. To investigate the mechanisms underlying this BP regulation, it is necessary to have a method to selectively modulate the activity of Purkinje cells (PCs), the sole output neurons from the cerebellar cortex, without affecting other neuronal types such as local interneurons or nonlocal neurons that send their axons to the cerebellar cortex. We recently developed a novel technique using optogenetics to manipulate PC activity and showed that activation and inhibition of PCs in the uvula either decreased or increased the resting BP, respectively. This technique was employed in the current study to examine the roles of the uvula in BP regulation during postural alterations in anesthetized rats. Enhanced Natronomonas pharaonis halorhodopsin (eNpHR), a light-driven chloride ion pump, was selectively expressed in uvular PCs using a lentiviral vector containing the PC-specific L7 promoter. The eNpHR-expressing PCs were then illuminated by orange laser (593 nm) either during 30° head-up or 30° head-down tilts. The eNpHR-mediated photoinhibition of the uvula attenuated the extent of BP recovery after a BP increase induced by postural changes during head-down tilts. By contrast, photoinhibition had no statistically significant effect on BP recovery during head-up tilts. The effects of photoinhibition on BP during tilts were significantly different from those observed during the resting condition, indicating that cerebellar control of BP during tilts is dynamic rather than static. Taken together, these results suggest that PCs in the uvula dynamically regulates BP maintenance during postural alterations.

Conversion of object identity to object-general semantic value in the primate temporal cortex

Faulty remembrance of objects past The primate brain analyzes visual input along the ventral processing stream to extract the identity of an object. The final stage of this stream, the perirhinal cortex, plays a crucial role in object recognition. Tamura et al. systematically biased the judgments of monkeys in an old-new object recognition task by using either optogenetic or electrical stimulation. The monkeys judged an encountered object as familiar when the stimulation site was in a hotspot where memory neurons were clustered. However, at the hotspots fringe region, where neurons lost selective responses to the learned objects, electrical microstimulation led the monkeys to mistakenly judge an object as never seen before. Science, this issue p. 687 Optogenetic activation of perirhinal memory neurons in primates drives a subjective feeling of having previously seen an object. At the final stage of the ventral visual stream, perirhinal neurons encode the identity of memorized objects through learning. However, it remains elusive whether and how object percepts alone, or concomitantly a nonphysical attribute of the objects (“learned”), are decoded from perirhinal activities. By combining monkey psychophysics with optogenetic and electrical stimulations, we found a focal spot of memory neurons where both stimulations led monkeys to preferentially judge presented objects as “already seen.” In an adjacent fringe area, where neurons did not exhibit selective responses to the learned objects, electrical stimulation induced the opposite behavioral bias toward “never seen before,” whereas optogenetic stimulation still induced bias toward “already seen.” These results suggest that mnemonic judgment of objects emerges via the decoding of their nonphysical attributes encoded by perirhinal neurons.

Optogenetics in the cerebellum: Purkinje cell-specific approaches for understanding local cerebellar functions.

The cerebellum consists of the cerebellar cortex and the cerebellar nuclei. Although the basic neuronal circuitry of the cerebellar cortex is uniform everywhere, anatomical data demonstrate that the input and output relationships of the cortex are spatially segregated between different cortical areas, which suggests that there are functional distinctions between these different areas. Perturbation of cerebellar cortical functions in a spatially restricted fashion is thus essential for investigating the distinctions among different cortical areas. In the cerebellar cortex, Purkinje cells are the sole output neurons that send information to downstream cerebellar and vestibular nuclei. Therefore, selective manipulation of Purkinje cell activities, without disturbing other neuronal types and passing fibers within the cortex, is a direct approach to spatially restrict the effects of perturbations. Although this type of approach has for many years been technically difficult, recent advances in optogenetics now enable selective activation or inhibition of Purkinje cell activities, with high temporal resolution. Here we discuss the effectiveness of using Purkinje cell-specific optogenetic approaches to elucidate the functions of local cerebellar cortex regions. We also discuss what improvements to current methods are necessary for future investigations of cerebellar functions to provide further advances.

Cofilin1 Controls Transcolumnar Plasticity in Dendritic Spines in Adult Barrel Cortex

During sensory deprivation, the barrel cortex undergoes expansion of a functional column representing spared inputs (spared column), into the neighboring deprived columns (representing deprived inputs) which are in turn shrunk. As a result, the neurons in a deprived column simultaneously increase and decrease their responses to spared and deprived inputs, respectively. Previous studies revealed that dendritic spines are remodeled during this barrel map plasticity. Because cofilin1, a predominant regulator of actin filament turnover, governs both the expansion and shrinkage of the dendritic spine structure in vitro, it hypothetically regulates both responses in barrel map plasticity. However, this hypothesis remains untested. Using lentiviral vectors, we knocked down cofilin1 locally within layer 2/3 neurons in a deprived column. Cofilin1-knocked-down neurons were optogenetically labeled using channelrhodopsin-2, and electrophysiological recordings were targeted to these knocked-down neurons. We showed that cofilin1 knockdown impaired response increases to spared inputs but preserved response decreases to deprived inputs, indicating that cofilin1 dependency is dissociated in these two types of barrel map plasticity. To explore the structural basis of this dissociation, we then analyzed spine densities on deprived column dendritic branches, which were supposed to receive dense horizontal transcolumnar projections from the spared column. We found that spine number increased in a cofilin1-dependent manner selectively in the distal part of the supragranular layer, where most of the transcolumnar projections existed. Our findings suggest that cofilin1-mediated actin dynamics regulate functional map plasticity in an input-specific manner through the dendritic spine remodeling that occurs in the horizontal transcolumnar circuits. These new mechanistic insights into transcolumnar plasticity in adult rats may have a general significance for understanding reorganization of neocortical circuits that have more sophisticated columnar organization than the rodent neocortex, such as the primate neocortex.

Avian sarcoma leukosis virus receptor-envelope system for simultaneous dissection of multiple neural circuits in mammalian brain

Significance Genetic dissection of multiple neural pathways remains challenging because of the limited number of genetic methods that can be used simultaneously. To overcome this limitation, we used modified avian sarcoma and leukosis virus envelopes and receptors to develop highly orthogonal genetic tools that can achieve expression of different genes in different target cells. From in vitro and in vivo screens, we identified tools that can specifically transfer genes of interest into mammalian neurons via engineered receptors, with minimal unintended interactions. Using this approach, we achieved pathway-specific, differential fluorescent labeling of three thalamic neuronal populations, each projecting into different cortical regions. Thus, our approach provides independent, simultaneous, and specific genetic tools for manipulating intermingled neural pathways in vivo. Pathway-specific gene delivery is requisite for understanding complex neuronal systems in which neurons that project to different target regions are locally intermingled. However, conventional genetic tools cannot achieve simultaneous, independent gene delivery into multiple target cells with high efficiency and low cross-reactivity. In this study, we systematically screened all receptor–envelope pairs resulting from the combination of four avian sarcoma leukosis virus (ASLV) envelopes (EnvA, EnvB, EnvC, and EnvE) and five engineered avian-derived receptors (TVA950, TVBS3, TVC, TVBT, and DR-46TVB) in vitro. Four of the 20 pairs exhibited both high infection rates (TVA–EnvA, 99.6%; TVBS3–EnvB, 97.7%; TVC–EnvC, 98.2%; and DR-46TVB–EnvE, 98.8%) and low cross-reactivity (<2.5%). Next, we tested these four receptor–envelope pairs in vivo in a pathway-specific gene-transfer method. Neurons projecting into a limited somatosensory area were labeled with each receptor by retrograde gene transfer. Three of the four pairs exhibited selective transduction into thalamocortical neurons expressing the paired receptor (>98%), with no observed cross-reaction. Finally, by expressing three receptor types in a single animal, we achieved pathway-specific, differential fluorescent labeling of three thalamic neuronal populations, each projecting into different somatosensory areas. Thus, we identified three orthogonal pairs from the list of ASLV subgroups and established a new vector system that provides a simultaneous, independent, and highly specific genetic tool for transferring genes into multiple target cells in vivo. Our approach is broadly applicable to pathway-specific labeling and functional analysis of diverse neuronal systems.

A new glass-coated tungsten optrode enclosing multiple optic fibers for fluorescence measurement, optogenetic photo-stimulation, and single-unit recording in deep brain regions

increasing the local electrical field of the observed region by increasing their resistance higher than around. The Ca imaging result indicated that the neurons not only on the electrode but ones around it responds. We found that the stimulating voltage where first Ca spikes occur has threshold. Also, we analyzed the Ca response by the raster plot of spikes, and confirmed specific Ca propagation between neurons. The trigger of the response is the Ca spike of on-electrode neurons. In conclusion, this device is the best tool to observe the change of the plasticity of neuronal synaptic connection by controlling stimulus timing of multiple electrode lines. Research fund: Strategic Research Foundation Grant-aided Project for Private Universities from Ministry of Education, Culture, Sport, Science, and Technology, Japan (MEXT), 2008–2012 (S0801008).