Sakiko Ujita

Development of a Far-Red to Near-Infrared Fluorescence Probe for Calcium Ion and its Application to Multicolor Neuronal Imaging

To improve optical imaging of Ca(2+) and to make available a distinct color window for multicolor imaging, we designed and synthesized CaSiR-1, a far-red to near-infrared fluorescence probe for Ca(2+), using Si-rhodamine (SiR) as the fluorophore and the well-known Ca(2+) chelator BAPTA. This wavelength region is advantageous, affording higher tissue penetration, lower background autofluorescence, and lower phototoxicity in comparison with the UV to visible range. CaSiR-1 has a high fluorescence off/on ratio of over 1000. We demonstrate its usefulness for multicolor fluorescence imaging of action potentials (visualized as increases in intracellular Ca(2+)) in brain slices loaded with sulforhodamine 101 (red color; specific for astrocytes) that were prepared from transgenic mice in which some neurons expressed green fluorescent protein.

Interpyramid Spike Transmission Stabilizes the Sparseness of Recurrent Network Activity

Cortical synaptic strengths vary substantially from synapse to synapse and exhibit a skewed distribution with a small fraction of synapses generating extremely large depolarizations. Using multiple whole-cell recordings from rat hippocampal CA3 pyramidal cells, we found that the amplitude of unitary excitatory postsynaptic conductances approximates a lognormal distribution and that in the presence of synaptic background noise, the strongest fraction of synapses could trigger action potentials in postsynaptic neurons even with single presynaptic action potentials, a phenomenon termed interpyramid spike transmission (IpST). The IpST probability reached 80%, depending on the network state. To examine how IpST impacts network dynamics, we simulated a recurrent neural network embedded with a few potent synapses. This network, unlike many classical neural networks, exhibited distinctive behaviors resembling cortical network activity in vivo. These behaviors included the following: 1) infrequent ongoing activity, 2) firing rates of individual neurons approximating a lognormal distribution, 3) asynchronous spikes among neurons, 4) net balance between excitation and inhibition, 5) network activity patterns that was robust against external perturbation, 6) responsiveness even to a single spike of a single excitatory neuron, and 7) precise firing sequences. Thus, IpST captures a surprising number of recent experimental findings in vivo. We propose that an unequally biased distribution with a few select strong synapses helps stabilize sparse neuronal activity, thereby reducing the total spiking cost, enhancing the circuit responsiveness, and ensuring reliable information transfer.

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.

Novelty-induced phase-locked firing to slow gamma oscillations in the hippocampus : requirement of synaptic plasticity

Temporally precise neuronal firing phase-locked to gamma oscillations is thought to mediate the dynamic interaction of neuronal populations, which is essential for information processing underlying higher-order functions such as learning and memory. However, the cellular mechanisms determining phase locking remain unclear. By devising a virus-mediated approach to perform multi-tetrode recording from genetically manipulated neurons, we demonstrated that synaptic plasticity dependent on the GluR1 subunit of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptor mediates two dynamic changes in neuronal firing in the hippocampal CA1 area during novel experiences: the establishment of phase-locked firing to slow gamma oscillations and the rapid formation of the spatial firing pattern of place cells. The results suggest a series of events potentially underlying the acquisition of new spatial information: slow gamma oscillations, originating from the CA3 area, induce the two GluR1-dependent changes of CA1 neuronal firing, which in turn determine information flow in the hippocampal-entorhinal system.

High‐Speed Multineuron Calcium Imaging Using Nipkow‐Type Confocal Microscopy

Conventional confocal and two‐photon microscopy scan the field of view sequentially with single‐point laser illumination. This raster‐scanning method constrains video speeds to tens of frames per second, which are too slow to capture the temporal patterns of fast electrical events initiated by neurons. Nipkow‐type spinning‐disk confocal microscopy resolves this problem by the use of multiple laser beams. We describe experimental procedures for functional multineuron calcium imaging (fMCI) based on Nipkow‐disk confocal microscopy, which enables us to monitor the activities of hundreds of neurons en masse at a cellular resolution at up to 2000 fps. Curr. Protoc. Neurosci. 57:2.14.1‐2.14.10.

Subtle modulation of ongoing calcium dynamics in astrocytic microdomains by sensory inputs

Astrocytes communicate with neurons through their processes. In vitro experiments have demonstrated that astrocytic processes exhibit calcium activity both spontaneously and in response to external stimuli; however, it has not been fully determined whether and how astrocytic subcellular domains respond to sensory input in vivo. We visualized the calcium signals in astrocytes in the primary visual cortex of awake, head‐fixed mice. Bias‐free analyses of two‐photon imaging data revealed that calcium activity prevailed in astrocytic subcellular domains, was coordinated with variable spot‐like patterns, and was dominantly spontaneous. Indeed, visual stimuli did not affect the frequency of calcium domain activity, but it increased the domain size, whereas tetrodotoxin reduced the sizes of spontaneous calcium domains and abolished their visual responses. The “evoked” domain activity exhibited no apparent orientation tuning and was distributed unevenly within the cell, constituting multiple active hotspots that were often also recruited in spontaneous activity. The hotspots existed dominantly in the somata and endfeet of astrocytes. Thus, the patterns of astrocytic calcium dynamics are intrinsically constrained and are subject to minor but significant modulation by sensory input.

cAMP-Dependent Calcium Oscillations of Astrocytes: An Implication for Pathology

Abstract Astrocytes in various brain regions exhibit spontaneous intracellular calcium elevations both in vitro and in vivo; however, neither the temporal pattern underlying this activity nor its function has been fully evaluated. Here, we utilized a long‐term optical imaging technique to analyze the calcium activity of more than 4000 astrocytes in acute hippocampal slices as well as in the neocortex and hippocampus of head‐restrained mice. Although astrocytic calcium activity was largely sparse and irregular, we observed a subset of cells in which the fluctuating calcium oscillations repeated at a regular interval of ˜30 s. These intermittent oscillations i) depended on type 2 inositol 1,4,5‐trisphosphate receptors; ii) consisted of a complex reverberatory interaction between the soma and processes of individual astrocytes; iii) did not synchronize with those of other astrocytes; iv) did not require neuronal firing; v) were modulated through cAMP‐protein kinase A signaling; vi) were facilitated under pathological conditions, such as energy deprivation and epileptiform hyperexcitation; and vii) were associated with enhanced hypertrophy in astrocytic processes, an early hallmark of reactive gliosis, which is observed in ischemia and epilepsy. Therefore, calcium oscillations appear to be associated with a pathological state in astrocytes.