Takuya Sasaki

Large-Scale Calcium Waves Traveling through Astrocytic Networks In Vivo

Macroscopic changes in cerebral blood flow, such as those captured by functional imaging of the brain, require highly organized, large-scale dynamics of astrocytes, glial cells that interact with both neuronal and cerebrovascular networks. However, astrocyte activity has been studied mainly at the level of individual cells, and information regarding their collective behavior is lacking. In this work, we monitored calcium activity simultaneously from hundreds of mouse hippocampal astrocytes in vivo and found that almost all astrocytes participated en masse in regenerative waves that propagated from cell to cell (referred to here as “glissandi”). Glissandi emerged depending on the neuronal activity and accompanied a reduction in infraslow fluctuations of local field potentials and a decrease in the flow of red blood cells. This novel phenomenon was heretofore overlooked, probably because of the high vulnerability of astrocytes to light damage; glissandi occurred only when observed at much lower laser intensities than previously used.

Action-Potential Modulation During Axonal Conduction

The waveform of an action potential can be physiologically modified while it travels along the axon. Once initiated near the soma, an action potential (AP) is thought to propagate autoregeneratively and distribute uniformly over axonal arbors. We challenge this classic view by showing that APs are subject to waveform modulation while they travel down axons. Using fluorescent patch-clamp pipettes, we recorded APs from axon branches of hippocampal CA3 pyramidal neurons ex vivo. The waveforms of axonal APs increased in width in response to the local application of glutamate and an adenosine A1 receptor antagonist to the axon shafts, but not to other unrelated axon branches. Uncaging of calcium in periaxonal astrocytes caused AP broadening through ionotropic glutamate receptor activation. The broadened APs triggered larger calcium elevations in presynaptic boutons and facilitated synaptic transmission to postsynaptic neurons. This local AP modification may enable axonal computation through the geometry of axon wiring.

Genetically encoded green fluorescent Ca2+ indicators with improved detectability for neuronal Ca2+ signals.

Imaging the activities of individual neurons with genetically encoded Ca2+ indicators (GECIs) is a promising method for understanding neuronal network functions. Here, we report GECIs with improved neuronal Ca2+ signal detectability, termed G-CaMP6 and G-CaMP8. Compared to a series of existing G-CaMPs, G-CaMP6 showed fairly high sensitivity and rapid kinetics, both of which are suitable properties for detecting subtle and fast neuronal activities. G-CaMP8 showed a greater signal (F max/F min = 38) than G-CaMP6 and demonstrated kinetics similar to those of G-CaMP6. Both GECIs could detect individual spikes from pyramidal neurons of cultured hippocampal slices or acute cortical slices with 100% detection rates, demonstrating their superior performance to existing GECIs. Because G-CaMP6 showed a higher sensitivity and brighter baseline fluorescence than G-CaMP8 in a cellular environment, we applied G-CaMP6 for Ca2+ imaging of dendritic spines, the putative postsynaptic sites. By expressing a G-CaMP6-actin fusion protein for the spines in hippocampal CA3 pyramidal neurons and electrically stimulating the granule cells of the dentate gyrus, which innervate CA3 pyramidal neurons, we found that sub-threshold stimulation triggered small Ca2+ responses in a limited number of spines with a low response rate in active spines, whereas supra-threshold stimulation triggered large fluorescence responses in virtually all of the spines with a 100% activity rate.

Circuit topology for synchronizing neurons in spontaneously active networks

Spike synchronization underlies information processing and storage in the brain. But how can neurons synchronize in a noisy network? By exploiting a high-speed (500–2,000 fps) multineuron imaging technique and a large-scale synapse mapping method, we directly compared spontaneous activity patterns and anatomical connectivity in hippocampal CA3 networks ex vivo. As compared to unconnected pairs, synaptically coupled neurons shared more common presynaptic neurons, received more correlated excitatory synaptic inputs, and emitted synchronized spikes with approximately 107 times higher probability. Importantly, common presynaptic parents per se synchronized more than unshared upstream neurons. Consistent with this, dynamic-clamp stimulation revealed that common inputs alone could not account for the realistic degree of synchronization unless presynaptic spikes synchronized among common parents. On a macroscopic scale, network activity was coordinated by a power-law scaling of synchronization, which engaged varying sets of densely interwired (thus highly synchronized) neuron groups. Thus, locally coherent activity converges on specific cell assemblies, thereby yielding complex ensemble dynamics. These segmentally synchronized pulse packets may serve as information modules that flow in associatively parallel network channels.

An Improved Genetically Encoded Red Fluorescent Ca2+ Indicator for Detecting Optically Evoked Action Potentials

Genetically encoded Ca2+ indicators (GECIs) are powerful tools to image activities of defined cell populations. Here, we developed an improved red fluorescent GECI, termed R-CaMP1.07, by mutagenizing R-GECO1. In HeLa cell assays, R-CaMP1.07 exhibited a 1.5–2-fold greater fluorescence response compared to R-GECO1. In hippocampal pyramidal neurons, R-CaMP1.07 detected Ca2+ transients triggered by single action potentials (APs) with a probability of 95% and a signal-to-noise ratio >7 at a frame rate of 50 Hz. The amplitudes of Ca2+ transients linearly correlated with the number of APs. The expression of R-CaMP1.07 did not significantly alter the electrophysiological properties or synaptic activity patterns. The co-expression of R-CaMP1.07 and channelrhodpsin-2 (ChR2), a photosensitive cation channel, in pyramidal neurons demonstrated that R-CaMP1.07 was applicable for the monitoring of Ca2+ transients in response to optically evoked APs, because the excitation light for R-CaMP1.07 hardly activated ChR2. These technical advancements provide a novel strategy for monitoring and manipulating neuronal activity with single cell resolution.

Expanding the repertoire of optogenetically targeted cells with an enhanced gene expression system.

Optogenetics has been enthusiastically pursued in recent neuroscience research, and the causal relationship between neural activity and behavior is becoming ever more accessible. Here, we established knockin-mediated enhanced gene expression by improved tetracycline-controlled gene induction (KENGE-tet) and succeeded in generating transgenic mice expressing a highly light-sensitive channelrhodopsin-2 mutant at levels sufficient to drive the activities of multiple cell types. This method requires two lines of mice: one that controls the pattern of expression and another that determines the protein to be produced. The generation of new lines of either type readily expands the repertoire to choose from. In addition to neurons, we were able to manipulate the activity of nonexcitable glial cells in vivo. This shows that our system is applicable not only to neuroscience but also to any biomedical study that requires understanding of how the activity of a selected population of cells propagates through the intricate organic systems.

Preinspiratory calcium rise in putative pre-Bötzinger complex astrocytes

•  Autonomic respiratory rhythm is essential to maintain lives and is generated in the lower brainstem. The ventrolateral medullary region, called the pre‐Bötzinger complex (preBötC), is the kernel for respiratory rhythm generation. Despite previous extensive studies focusing on neurons, the mechanism of how respiratory rhythm is generated has not been fully understood. •  Here we show that non‐neuronal glial cells (a subset of putative astrocytes) in the preBötC are periodically activated preceding inspiratory neuronal activity, periodic activity of putative astrocytes persists during blockade of neuronal activity, and stimulation of astrocytes in the preBötC induces inspiratory neuronal firings. •  These findings together with the previous report that blockade of astrocytic metabolism abolishes inspiratory neural output suggest that astrocytes are functionally involved in respiratory rhythm generation. •  These results will help us better understand how respiratory rhythm is generated and how respiratory output is disturbed in various pathological conditions.

Effects of Axonal Topology on the Somatic Modulation of Synaptic Outputs

Depolarization of the neuronal soma augments synaptic output onto postsynaptic neurons via long-range, axonal cable properties. Here, we report that the range of this somatic influence is spatially restricted by not only axonal path length but also a branching-dependent decrease in axon diameter. Cell-attached recordings of action potentials (APs) from multiple axon branches of a rat hippocampal CA3 pyramidal cell revealed that an AP was broadened following a 20 mV depolarization of the soma and reverted to a normal width during propagation down the axon. The narrowing of the AP depended on the distance traveled by the AP and on the number of axon branch points through which the AP passed. These findings were confirmed by optical imaging of AP-induced calcium elevations in presynaptic boutons, suggesting that the somatic membrane potential modifies synaptic outputs near the soma but not long-projection outputs. Consistent with this prediction, whole-cell recordings from synaptically connected neurons revealed that depolarization of presynaptic CA3 pyramidal cells facilitated synaptic transmission to nearby CA3 pyramidal cells, but not to distant pyramidal cells in CA3 or CA1. Therefore, axonal geometry enables the differential modulation of synaptic output depending on target location.

Targeted axon-attached recording with fluorescent patch-clamp pipettes in brain slices

Understanding the physiology of axons in the central nervous system requires experimental access to intact axons. This protocol describes how to perform cell-attached recordings from narrow axon fibers (ϕ <1 μm) in acute and cultured brain slice preparations (with a success rate of ∼50%). By using fluorophore-coated glass pipettes and Nipkow disk confocal microscopy, fluorescently labeled axons can be visually targeted under online optical control. In the cell-attached configuration, axonal action potentials are extracellularly recorded as unit-like, sharp negative currents. The axon morphology labeling and cell-attached recordings of axons can be completed within 1–2 h. The recordings are stable for at least 30 min.

Heterogeneity and independency of unitary synaptic outputs from hippocampal CA3 pyramidal cells.

•  Excitatory neurotransmission in the cortex is a local event that occurs at chemical synaptic junctions, but it has not been experimentally addressed at the large network scale. •  Here we show that trial‐to‐trial variability in the synaptic transmission at hippocampal synapses is attributable to fluctuations in calcium elevations in axonal boutons, rather than to changes in the action potential waveform in axons. •  Multiple patch‐clamp recordings from synaptically connected neural circuits revealed no correlation between synaptic outputs to two different postsynaptic cells from a common presynaptic cell. •  This independency of synapses was likely to arise from unique calcium dynamics within presynaptic terminals.