Imaging the brain: advanced optical methods to investigate neural functions

Editors

Jonathan Mapelli

University of Modena and Reggio Emilia

Egidio D‘Angelo

University of Pavia

Impact

Mini Review

09 May 2017

New Tools to Study Astrocyte Ca2+ Signal Dynamics in Brain Networks In Vivo

Gabriele Losi

, 2 more and

Giorgio Carmignoto

Sensory information processing is a fundamental operation in the brain that is based on dynamic interactions between different neuronal populations. Astrocytes, a type of glial cells, have been proposed to represent active elements of brain microcircuits that, through dynamic interactions with neurons, provide a modulatory control of neuronal network activity. Specifically, astrocytes in different brain regions have been described to respond to neuronal signals with intracellular Ca²⁺ elevations that represent a key step in the functional recruitment of astrocytes to specific brain circuits. Accumulating evidence shows that Ca²⁺ elevations regulate the release of gliotransmitters that, in turn, modulate synaptic transmission and neuronal excitability. Recent studies also provided new insights into the spatial and temporal features of astrocytic Ca²⁺ elevations revealing a surprising complexity of Ca²⁺ signal dynamics in astrocytes. Here we discuss how recently developed experimental tools such as the genetically encoded Ca²⁺ indicators (GECI), optogenetics and chemogenetics can be applied to the study of astrocytic Ca²⁺ signals in the living brain.

10,884 views

30 citations

Optimal 2P photolysis of caged glutamate with wavefront correction. (a,b) Representative 2P flattened stack images of neurons showing location of the dendritic spine targeted for 2P photolysis. Average light evoked EPSP responses (black traces) each superimposed on ten single episodes (gray traces) measured in a layer 5 pyramidal cell, showing example of wavefront correction before (top panel) and after (bottom panel) for (c) astigmatism, (d) coma-x, (e) spherical aberration, and (f) coma-y. Scale bar 20 μm.

Original Research

01 May 2017

Improving Focal Photostimulation of Cortical Neurons with Pre-derived Wavefront Correction

Julian M. C. Choy

, 4 more and

Vincent R. Daria

2,625 views

6 citations

Original Research

19 April 2017

Correlative STED and Atomic Force Microscopy on Live Astrocytes Reveals Plasticity of Cytoskeletal Structure and Membrane Physical Properties during Polarized Migration

Nathan Curry

, 3 more and

Clemens F. Kaminski

The plasticity of the cytoskeleton architecture and membrane properties is important for the establishment of cell polarity, adhesion and migration. Here, we present a method which combines stimulated emission depletion (STED) super-resolution imaging and atomic force microscopy (AFM) to correlate cytoskeletal structural information with membrane physical properties in live astrocytes. Using STED compatible dyes for live cell imaging of the cytoskeleton, and simultaneously mapping the cell surface topology with AFM, we obtain unprecedented detail of highly organized networks of actin and microtubules in astrocytes. Combining mechanical data from AFM with optical imaging of actin and tubulin further reveals links between cytoskeleton organization and membrane properties. Using this methodology we illustrate that scratch-induced migration induces cytoskeleton remodeling. The latter is caused by a polarization of actin and microtubule elements within astroglial cell processes, which correlates strongly with changes in cell stiffness. The method opens new avenues for the dynamic probing of the membrane structural and functional plasticity of living brain cells. It is a powerful tool for providing new insights into mechanisms of cell structural remodeling during physiological or pathological processes, such as brain development or tumorigenesis.

8,684 views

56 citations

Conditions to evoke a train of action potentials in neurons of mouse visual cortex Layer 2/3 with a fiber amplified Laser System at 1030 nm. (A) Trains of 2P holographic stimulation pulses were used to evoke trains of APs in single L2/3 cells: example of a pyramidal cell. Trains of 10 pulses at frequencies ranging from 5 to 40 Hz and LPDs at AP threshold were used. (B) Same as (A) for a fast spiking cell. (C) Plot of the maximum AP frequency vs. the photo-stimulation light pulses frequency, for pyramidal cells and LTS cells (red triangles, average of n = 19 cells) and FS cells (green diamonds, average of n = 4 cells). Black line corresponds to an AP frequency equal to the light pulse frequency. (D) Maximum AP frequency reached for trains of a given number of light pulses for pyramidal cells and LTS cells (red triangles, average of n = 19 cells) and FS cells (green diamonds, average of n = 4 cells).

Original Research

18 October 2016

Two-Photon Holographic Stimulation of ReaChR

Emmanuelle Chaigneau

, 7 more and

Valentina Emiliani

10,829 views

76 citations

Effects of neurosteroids on SR101 uptake in the hippocampus. (A) Astroglial EGFP-fluorescence (green) and SR101-fluorescence (red) in the hippocampus after a staining in control conditions (1 μM SR101 for 20 min at 34°C). (B) Reduced SR101-labeling of hippocampal astrocytes when the neurosteroid allopregnanolone sulfate (APAS, 100 μM) was included in the staining solution. Both figures show maximum intensity projection 51 stacks (2 μm distance). (C–F) Images from single layer for EGFP (C,D) and SR101 (E,F). Note the reduction of the intensity of SR101 after application of APAS (F). (G–I) Statistical analysis: (G) The reduction of the SR101-intensity by 100 μM APAS is significant (Mann-Whitney Rank Sum Test; p = 0.032; n = 5 slices; 3 mice). (H) Additionally the number of SR101 positive cells than can be identified after APAS is reduced (t-Test; p = 0.040). (I) The APAS-induced reduction of SR101 intensity in EGFP-positive cells and EGFP-negative SR101 labeled cells is not different (Mann-Whitney Rank Sum Test; p = 0.548). Asterisks in (G,H) indicate significance (p < 0.05).

Hypothesis and Theory

28 February 2017

Limitations of Sulforhodamine 101 for Brain Imaging

Swen Hülsmann

, 2 more and

Christian Schnell

8,833 views

32 citations

Original Research

14 April 2016

General Anesthetic Conditions Induce Network Synchrony and Disrupt Sensory Processing in the Cortex

Thomas Lissek

, 5 more and

Mazahir T. Hasan

Deep anesthesia enhances neural synchrony. (A) Random example fluorescence traces of the same cell under different anesthetic conditions. Vertical bars mark detected peaks, inset: 5% ΔR/R (y-axis), 5 s (x-axis). (B) Extracted peak time points of all cells shown as probabilities over adjacent frames (Top panel, scale bar: 5 s), color-coded correlation matrices for Pearson's r of 3 example trials (Bottom panel, small pictures) and example correlation maps (Bottom panel, big pictures, scale bar: 30 μm). Inset: color-code for peak probability (white = 0 to black = 1). Dotted lines indicate time points of whisker stimulation. Correlation is expressed as Pearson's correlation coefficient (r) and color-coded from r = 0 (blue, no correlation) to r = 1 (red, perfect positive correlation). (C) The number of detected peaks per cell (mean over all trials and cells ± SD; ANOVA, p = 0.71). (D) Correlation r's filtered for significance (p < 0.05) shift toward higher values with high isoflurane: cumulative frequency distribution for all pairwise correlation r's (p < 0.01 High Iso vs. Low Iso). (E) High isoflurane concentrations induce a marked increase in overall synchrony that is independent of sensory stimulation. (Left) Mean pairwise correlation of all cell pairs for the indicated isoflurane concentration over all trials per mouse (mean ± SEM, *p < 0.05, **p < 0.01 ***p < 0.001). (Right) Summary of correlation r-values under a high isoflurane concentration (mean ± SEM). The same analysis as above was additionally performed for trials where only auditory stimulation was present (All peaks vs. subtracted p = 0.58, All peaks vs. Sound only; p = 0.43). (F) High isoflurane concentrations preferentially increase synchrony between nearby neurons. Shown is the mean pairwise correlation (Pearson's r) plotted against pairwise distance (bin size: 25 μm) (mean ± SEM).

General anesthetics are commonly used in animal models to study how sensory signals are represented in the brain. Here, we used two-photon (2P) calcium activity imaging with cellular resolution to investigate how neuronal activity in layer 2/3 of the mouse barrel cortex is modified under the influence of different concentrations of chemically distinct general anesthetics. Our results show that a high isoflurane dose induces synchrony in local neuronal networks and these cortical activity patterns closely resemble those observed in EEG recordings under deep anesthesia. Moreover, ketamine and urethane also induced similar activity patterns. While investigating the effects of deep isoflurane anesthesia on whisker and auditory evoked responses in the barrel cortex, we found that dedicated spatial regions for sensory signal processing become disrupted. We propose that our isoflurane-2P imaging paradigm can serve as an attractive model system to dissect cellular and molecular mechanisms that induce the anesthetic state, and it might also provide important insight into sleep-like brain states and consciousness.

11,692 views

34 citations

Cumulated results demonstrating the equalization of the peak current by graded intensity photostimulation. The ratio between peak currents evoked with illumination of equal intensity is compared to the case of intensity-graded illumination (A) for n = 9 pairs of differently expressing cells (where cell_1 is the more expressing one) (avg. ratio of equal stimulation peak currents = 4 ± 2; avg. ratio upon graded stimulation = 1.3 ± 0.7). (B) Plot of the corresponding ratio of peak current vs the ratio of fluorescence detected for all couples of cells. Superposed in black the identity line (y = x). The two pairs of cells shown in green in the plots are cells in which the ratio between fluorescence and currents did not correspond and for which the protocol gave a less good equalization.

Methods

17 October 2016

Computer Generated Holography with Intensity-Graded Patterns

Rossella Conti

, 3 more and

Valentina Emiliani

5,109 views

18 citations

Methods

06 January 2016

In vivo Calcium Imaging of Evoked Calcium Waves in the Embryonic Cortex

Mikhail Yuryev

, 5 more and

Claudio Rivera

The dynamics of intracellular calcium fluxes are instrumental in the proliferation, differentiation, and migration of neuronal cells. Knowledge thus far of the relationship between these calcium changes and physiological processes in the developing brain has derived principally from ex vivo and in vitro experiments. Here, we present a new method to image intracellular calcium flux in the cerebral cortex of live rodent embryos, whilst attached to the dam through the umbilical cord. Using this approach we demonstrate induction of calcium waves by laser stimulation. These waves are sensitive to ATP-receptor blockade and are significantly increased by pharmacological facilitation of intracellular-calcium release. This approach is the closest to physiological conditions yet achieved for imaging of calcium in the embryonic brain and as such opens new avenues for the study of prenatal brain development. Furthermore, the developed method could open the possibilities of preclinical translational studies in embryos particularly important for developmentally related diseases such as schizophrenia and autism.

8,592 views

22 citations

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