Multicellular glial calcium waves may locally regulate neural activity or brain energetics. Here, we report a diffusion-driven astrocytic signal in the normal, intact brain that spans many astrocytic processes in a confined volume without fully encompassing any one cell. By using 2-photon microscopy in rodent cerebellar cortex labeled with fluorescent indicator dyes or the calcium-sensor protein G-CaMP2, we discovered spontaneous calcium waves that filled approximately ellipsoidal domains of Bergmann glia processes. Waves spread in 3 dimensions at a speed of 4 -11 m/s to a diameter of Ϸ50 m, slowed during expansion, and were reversibly blocked by P2 receptor antagonists. Consistent with the hypothesis that ATP acts as a diffusible trigger of calcium release waves, local ejection of ATP triggered P2 receptor-mediated waves that were refractory to repeated activation. Transglial waves represent a means for purinergic signals to act with local specificity to modulate activity or energetics in local neural circuits.astrocytes ͉ Bergmann glia ͉ in vivo ͉ 2-photon microscopy ͉ G-CaMP2

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 detect discrete 1−2 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 5–10 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.

Sensitivity spectra of Stark-shift voltage sensitive dyes, such as ANNINE-6, suggest the use of the extreme red edges of the excitation spectrum to achieve large fractional fluorescence changes with membrane voltage. This was tested in cultured HEK293 cells. Cells were illuminated with light at the very red edge of the dye's excitation spectrum, where the absorption cross section is as much as 100 times smaller than at its peak. The small-signal fractional fluorescence changes were -0.17%/mV, -0.28%/mV, and -0.35%/mV for one-photon excitation at 458 nm, 488 nm, and 514 nm, respectively, and -0.29%/mV, -0.43%/mV, and -0.52%/mV for two-photon excitation at 960 nm, 1000 nm, and 1040 nm, respectively. For large voltage swings the fluorescence changes became nonlinear, reaching 50% and -28% for 100 mV hyper- and depolarization, respectively, at 514 nm and 70% and -40% at 1040 nm. Such fractional sensitivities are approximately 5 times larger than what is commonly found with other voltage-sensing dyes and approach the theoretical limit given by the spectral Boltzmann tail.

The voltage sensitivity of hemicyanine dyes ANNINE-6 and ANNINE-5 with anellated benzene rings and without free CC single and double bonds is studied in Retzius neurons from Hirudo medicinalis. For comparison, biaryl hemicyanine BNBIQ and styryl hemicyanines di-4-ANEPBS and RH-421 are investigated. Fluorescence spectra are recorded by an independent variation of the wavelengths of excitation and emission at two defined membrane voltages. With extracellular staining, a positive change in the intracellular voltage shifts all excitation spectra to the blue. That modulation is assigned to a molecular Stark effect that increases in the series RH-421, di-4-ANEPBS, BNBIQ, ANNINE-5, and ANNINE-6 with displacements of elementary charge by 0.24, 0.43, 0.51, 0.65, and 0.81 nm across the membrane. For BNBIQ, ANNINE-5, and ANNINE-6, an almost identical blue shift is observed for the emission that is also assigned to a Stark effect. The ANNINE dyes are the most efficient fluorescent probes of neuronal activity on the basis of a well-defined physical mechanism. The implications with respect to the optical recording of voltage transients are considered.

ET DWI is more effective for decreasing regional variability of ADC and IVIM parameters than FB DWI or RT DWI; it may improve measurement repeatability by reducing cardiac motion-induced measurement error.

Conventional methods of imaging membrane potential changes have limited spatial resolution, particularly along the axis perpendicular to the cortical surface. The laminar organization of the cortex suggests, however, that the distribution of activity in depth is not uniform. We developed a technique to resolve network activity of different cortical layers in vivo using two-photon microscopy of the voltage-sensitive dye (VSD) ANNINE-6. We imaged spontaneous voltage changes in the barrel field of the somatosensory cortex of head-restrained mice and analyzed their spatiotemporal correlations during anesthesia and wakefulness. EEG recordings always correlated more strongly with VSD signals in layer (L) 2 than in L1. Nearby (<200 m) cortical areas were correlated with one another during anesthesia. Waking the mouse strongly desynchronized neighboring cortical areas in L1 in the 4-to 10-Hz frequency band. Wakefulness also slightly increased synchrony of neighboring territories in L2 in the 0.5-to 4.0-Hz range. Our observations are consistent with the idea that, in the awake animal, long-range inputs to L1 of the sensory cortex from various cortical and thalamic areas exert top-down control on sensory processing.ayer (L) 1 has been least investigated of all the cortical layers despite its potentially prominent role in cortical processing. Although largely acellular, except for a sparse population of inhibitory neurons (1), L1 is the main target of axons originating in distant cortical and subcortical regions. This dense plexus of axons makes excitatory synapses on apical dendritic tufts of L2/3 and L5 pyramidal neurons. Dendritic tufts contain powerful active conductances that cause electrical changes that can propagate to their relatively distant somata and influence the integration of synaptic inputs there (2). L1 is therefore ideally positioned to mediate communication between brain regions.In the primary somatosensory cortex, long-range input to L1 consists of corticocortical synapses from primary motor cortex and the secondary somatosensory area (3). In contrast, synapses in other cortical layers, such as L2, originate from a mixture of excitatory and inhibitory cells located mainly in that specific cortical column (4). A major source of thalamocortical synapses in L1 is the second-order sensory nucleus, also called the ''posterior medial'' (POm) nucleus (5), whose activity appears to be strongly modulated by arousal (6). This finding suggests that synaptic inputs in L1 also should be modulated by arousal. Synaptic inputs can be studied by recording membrane-voltage (V m ) changes, but L1 is relatively inaccessible using conventional methods.Wide-field imaging of voltage-sensitive dyes (VSDs) has been used successfully to sample V m in neurites of isolated neurons and across large populations of cortical cells (7). Although such imaging allows high frame rates (Ͼ1 kHz), scattering and limited depth discrimination result in poor spatial resolution. Thus, when imaging in vivo, the signal at any point in the image co...

We present a novel voltage-sensitive hemicyanine dye ANNINE-6plus and describe its synthesis, its properties and its voltage-sensitivity in neurons. The dye ANNINE-6plus is a salt with a double positively charged chromophore and two bromide counterions. It is derived from the zwitterionic dye ANNINE-6. While ANNINE-6 is insoluble in water, ANNINE-6plus exhibits a high solubility of around 1 mM. Nonetheless, it displays a strong binding to lipid membranes. In contrast to AN-NINE-6, the novel dye can be used to stain cells from aqueous solution without surfactants or organic solvents. In neuronal membranes, ANNINE-6plus exhibits the same molecular Stark effect as ANNINE-6. As a consequence, a high voltage-sensitivity is achieved with illumination and detection in the red end of the excitation and emission spectra, respectively. ANNINE-6plus will be particularly useful for sensitive optical recording of neuronal excitation when organic solvents and surfactants must be avoided as with intracellular or extracellular staining of brain tissue.

Chronic cranial windows have been instrumental in advancing optical studies in vivo, permitting long-term, high-resolution imaging in various brain regions. However, once a window is attached it is difficult to regain access to the brain under the window for cellular manipulations. Here we describe a simple device that combines long term in vivo optical imaging with direct brain access via glass or quartz pipettes and metal, glass, or quartz electrodes for cellular manipulations like dye or drug injections and electrophysiological stimulations or recordings while keeping the craniotomy sterile. Our device comprises a regular cranial window glass coverslip with a drilled access hole later sealed with biocompatible silicone. This chronic cranial window with access port is cheap, easy to manufacture, can be mounted just as the regular chronic cranial window, and is self-sealing after retraction of the pipette or electrode. We demonstrate that multiple injections can be performed through the silicone port by repetitively bolus loading calcium sensitive dye into mouse barrel cortex and recording spontaneous cellular activity over a period of weeks. As an example to the extent of its utility for electrophysiological recording, we describe how simple removal of the silicone seal can permit patch pipette access for whole-cell patch clamp recordings in vivo. During these chronic experiments we do not observe any infections under the window or impairment of animal health.