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Near-Infrared Carbon Nanotube Tracking Reveals the Nanoscale Extracellular Space around Synapses
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  • Chiara Paviolo
    Chiara Paviolo
    Université de Bordeaux, Institut d’Optique & Centre National de la Recherche Scientifique, UMR 5298, 33400 Talence, France
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  • Joana S. Ferreira
    Joana S. Ferreira
    Université de Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, 33076 Bordeaux, France
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  • Antony Lee
    Antony Lee
    Université de Bordeaux, Institut d’Optique & Centre National de la Recherche Scientifique, UMR 5298, 33400 Talence, France
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    Orcidhttps://orcid.org/0000-0003-2193-5369
  • Daniel Hunter
    Daniel Hunter
    Université de Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, 33076 Bordeaux, France
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  • Ivo Calaresu
    Ivo Calaresu
    Université de Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, 33076 Bordeaux, France
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  • Somen Nandi
    Somen Nandi
    Université de Bordeaux, Institut d’Optique & Centre National de la Recherche Scientifique, UMR 5298, 33400 Talence, France
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  • Laurent Groc*
    Laurent Groc
    Université de Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, 33076 Bordeaux, France
    *Email: [email protected]
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  • Laurent Cognet*
    Laurent Cognet
    Université de Bordeaux, Institut d’Optique & Centre National de la Recherche Scientifique, UMR 5298, 33400 Talence, France
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0002-3573-5387
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Nano Letters

Cite this: Nano Lett. 2022, 22, 17, 6849–6856
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https://doi.org/10.1021/acs.nanolett.1c04259
Published August 29, 2022

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Abstract

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We provide evidence of a local synaptic nanoenvironment in the brain extracellular space (ECS) lying within 500 nm of postsynaptic densities. To reveal this brain compartment, we developed a correlative imaging approach dedicated to thick brain tissue based on single-particle tracking of individual fluorescent single wall carbon nanotubes (SWCNTs) in living samples and on speckle-based HiLo microscopy of synaptic labels. We show that the extracellular space around synapses bears specific properties in terms of morphology at the nanoscale and inner diffusivity. We finally show that the ECS juxta-synaptic region changes its diffusion parameters in response to neuronal activity, indicating that this nanoenvironment might play a role in the regulation of brain activity.

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Neuronal communication in the central nervous system mainly occurs at the level of synapses through the release of neurotransmitters in the synaptic cleft and the activation of postsynaptic receptors. Neurotransmitters can spill over from synapses and act at a distance through a process known as “volume transmission”, in which signaling molecules navigate within the brain extracellular space (ECS). (1,2) Despite technical and molecular advances over the past decades, the local dimensions and architecture of this complex environment have yet to be elucidated in identified regions of the living brain. In particular, the design and combination of experimental strategies offering nanoscale resolution and sectioning capabilities are needed to correlate the narrow and tortuous environment with specific cellular structures. (3)

Changes in the ECS can affect neuronal excitability and signal transmission by altering local ion concentrations in the healthy and diseased brain. (4−6) Compared with free diffusion in an “open space” where molecules move randomly, diffusion in the ECS is critically dependent on the physical and chemical structure of the local microenvironment. (7) Zheng et al. investigated the extracellular diffusivity inside the cleft of synapses formed by hippocampal mossy fibers which could be resolved by diffraction limited microscopy approaches, suggesting reduced diffusion when compared to free medium. (8) Yet, understanding the mechanisms through which the ECS modulates neuronal communication, particularly around excitatory synapses at the nanoscale, still represents a key challenge in brain research. (3) Because the characteristics of the ECS around synapses remain unknown at the submicron scale in native live brain tissue, it is important to establish whether the synaptic ECS environment displays specific dimensions and diffusional properties.

To tackle this question, we developed a correlative imaging approach based on single-particle tracking of individual fluorescent single wall carbon nanotubes (SWCNTs) in living brain tissues and on speckle-based microscopy of synaptic labels (Figure 1A). The first approach provides information about ECS dimension and diffusion properties at the nanoscale, while the second one allows us to identify and localize postsynaptic densities inside brain tissues. Using this method, we revealed the existence of a local synaptic nanoenvironment (which we called “juxta-synaptic” region) lying within 500 nm of the postsynaptic densities (PSDs). This local ECS environment is defined by specific properties in terms of morphology at the nanoscale and inner diffusivity. We finally show that the juxta-synaptic region changes its diffusion parameters in response to neuronal activity, indicating that its features might play a role in the regulation of brain activity.

Figure 1

Figure 1. Experimental conditions. (A) Schematic of the experimental setup. Imaging was performed on a customized epifluorescent microscope equipped with a speckle based illumination setup. NIR imaging of SWCNTs was performed at 845 nm with a 60× immersion objective. GFP-PSD95 expressing neurons were imaged at 488 nm through the same objective and recorded by a HiLo modality. Individual SWCNTs were followed in the ECS of living brains using an EM-CCD camera. A standard white light fiber and low magnification objective (4×) were initially used to check the CA1 position in the hippocampal slice. (B) Schematic of the organotypic slices. Rat organotypic cultures were prepared from rat pup brains and cultured for 4 days in vitro (DIV) before PSD95 infection. SWCNTs were incubated at DIV 12–14 for 2 h. (C) Example of a speckle and HiLo images recorded with the Sparq system. Individual GFP-PSD95 clusters were clearly recognizable. Scale bars are 20 μm. (D) Typical recording of SWCNTs in living organotypic slices. Nanotubes were sparsely and individually detected at high signal-to-noise ratio. Scale bar is 20 μm.

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Near-infrared luminescent SWCNTs are interesting nanoparticles for deep brain tissue microscopy, (9) due to their unique brightness, photostability, and near-infrared emission range in the biological window. (10) SWCNTs also demonstrated the capability to locally probe in situ chemical species, including neurotransmitters. (11) At the single nanotube level, we have recently shown that SWCNTs can access ECS in the brain tissue of young (12) and adult (13) rodents, and their diffusion trajectories can reveal ECS local dimensions at the nanoscale. It is noteworthy that another optical approach based on STED microscopy (14) could provide similar structural ECS dimensions. Using these optical methodologies, ECS remodeling in a neuropathological condition was successfully reported. (14,15) Furthermore, contrary to other microscopy techniques displaying nanoscale resolution for the study of structural tissue features (also including electron microscopy), SWCNT tracking allows us to investigate ECS architectures in intact living brain at unrivaled depths (>10 μm), and in addition, it has the unique ability to reveal diffusion properties inside the tissues. To locally probe the ECS dimension and diffusivity around synapses deep inside living brain tissues, the use of SWCNTs thus emerged as the tool of choice.

In order to identify synaptic areas into cultured hippocampal brain slices (Figure S1), we fluorescently labeled synapses using GFP-PSD95 lentivirus vectors (Figures 1 and S2). PSD95 is one of the most abundant proteins in excitatory synapses and is a common marker of postsynaptic areas. Synaptic imaging was performed in the CA1 region of the hippocampus at depth ranging from 10 to approximately 50 μm (Figure S3A) using a speckle based structured illumination technique known as HiLo microscopy. (16) HiLo is a wide-field fluorescence microscopy method which provides excellent optical sectioning capabilities in thick biological samples, akin to confocal microscopy but at higher imaging rates and with simpler instrumentation. It requires the acquisition of two images: one using a uniform illumination and one with structured illumination (here the illumination transported through a multimode fiber is randomly structured by the speckle). These two images are used to extract the high and low frequency in-focus contents (from here, the acronym HiLo), leading to a full resolution in-focus image containing the entire frequency bandwidth of the imaging system (Figure 1C). The basic principle is that the in-focus high frequency content of the image can be extracted by high-pass filtering of the image acquired using uniform illumination, while the in-focus low frequency content can be obtained from contrast analysis of the image obtained with structured illumination. (17) From the HiLo images, GFP positive clusters corresponding to synapses were then identified in living brain tissues (Figure 1C).

Biocompatible fluorescent SWCNTs were prepared by encapsulation with phospholipid–polyethylene glycol (PL–PEG) molecules. This coating minimizes nonspecific adsorption onto biological structures (18) while preserving SWCNT luminescence brightness for single molecule experiments. Cultured hippocampal slices expressing GFP-PSD95 were incubated with PL–PEG coated SWCNTs, and slices were placed onto an NIR single molecule microscope (see Material and Methods). We focused on (6,5) SWCNTs emitting at 985 nm which are efficiently excited at 845 nm while minimizing light absorption by the tissue. (19) Bright (6,5) SWCNTs were sparsely and individually detected at high signal-to-noise ratio with low autofluorescence (coming from biological structures or from out-of-focus nanotubes (Figure 1D), which constitutes a decisive asset to perform single molecule imaging at the required depth. Indeed, investigating relevant ECS structures inherently requires thick brain tissue preparations (generally a few hundred micrometers). Luminescent SWCNTs were imaged at 33 frames per second to grasp their rapid diffusion within the ECS (see Movie S1). Importantly, the SWCNT high aspect ratio and intrinsic rigidity play a decisive role here, slowing down nanotube diffusion in the ECS maze while ensuring high accessibility to nanoscale environments. (20) These are unique features of these bright non-photobleaching 1D nanoparticles.

Superlocalization analysis of nanotube positions along their trajectories was performed as follows. For each recorded movie frame, we applied a 2D asymmetric Gaussian fitting analysis of the fluorescence profiles to decipher the nanotube centroids with subwavelength precision (∼50 nm) and nanotube length (long axis of the Gaussian fit). Taking into account the exciton diffusion range that decreases the apparent nanotube length in fluorescence images due to end quenching, (21) the measured nanotube length distribution was centered around ∼600 nm (Figure S3B). This narrow distribution confirms the consistency and reproducibility of the nanotube preparation over 14 independent experiments (3 slices per experiment; 76 analyzed SWCNTs).

In order to explore the ECS environment around the synapses, the region around each GFP-PSD95 centroid was segmented by a series of concentric coronal areas of 100 nm widths (Figure 2A). A maximum distance of 1 μm from the synaptic centroids was considered, based on the average synapse density in hippocampal neurons (i.e., around 1 spine per μm of dendrite (22)). An image correlation analysis with SWCNT localizations allowed us to create a distance-to-synapse investigation of the ECS features. We first analyzed SWCNT local diffusivity in the different coronal areas. Local diffusivity was defined as the normalized local instantaneous diffusion along trajectories. For this, the two-dimensional mean-squared displacement (MSD) was calculated as a function of time intervals along a sliding window of 390 ms for each trajectory. By approximating the MSD as linear at short time intervals, a linear fit of the first 90 ms yielded Dinst, the instantaneous diffusion coefficient. The normalized diffusivity was then obtained by calculating Dinst/Dref where Dref is the calculated diffusion coefficient of carbon nanotubes freely diffusing in a fluid having the viscosity ηref of the cerebrospinal fluid (CSF) (7) (see Material and Methods).

Figure 2

Figure 2. (A) Schematic of the analysis. Individual SWCNT trajectories were analyzed on coronal areas of 100 nm from the synaptic centroids. Scale bar is 1 μm. (B) Median (red crosses), 25th/75th percentile (shaded area), and cumulative distribution functions (inset) of the diffusivity values calculated at each coronal area for control conditions (31 SWCNTs). A sharp transition in the diffusivity values was observed between 400 and 500 nm from the PSD95 centroids.

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Figure 2B shows median values and cumulative distributions (inset) of local diffusivities measured in different coronal areas around synapses. Clearly, a distance-to-synapse dependent behavior lies within submicron scales (Figure 2B). More specifically, a sharp transition in the diffusivity is observed between 400 and 500 nm revealing a specific diffusivity behavior around synapses where the particles undergo a 10-fold enhanced diffusivity as compared to farther away from synapses. In order to unambiguously assess the presence of this specific juxta-synaptic diffusion environment, a series of controls was performed. First, we simulated SWCNT trajectories assuming Brownian motion and randomly generated synaptic localizations to rule out that the generation of 100-nm-width coronal regions might bias apparent diffusivities into reduced coronal areas (see Material and Methods and Figure S4A,B. Furthermore, using the experimental SWCNT trajectories, we also generated “fake” (randomized) localizations of synapses in regions where no positive GFP-PSD95 signals were experimentally identified: no specific (enhanced) diffusivities were subsequently generated around the “fake” synaptic areas (Figure S4C). We thus conclude that a “juxta-synaptic” environment exists up to 500 nm away from synaptic centroids where the diffusivities are larger than in “non-juxta-synaptic” areas (areas between 500 and 1000 nm away from GFP-PSD95 centroids). We finally concluded that the presence of nanotubes in the juxta-synaptic regions did not alter synaptic spontaneous activity. For this, dissociated primary neuronal cultures were infected with GCaMP6, a calcium reporter, to monitor the spontaneous activity of excitatory synapses following exposure to SWCNTs (Figure S5A). For this control, we deliberately used a particle concentration resulting in imaged densities (∼4 × 1010 SWCNT.mL–1) far exceeding those observed in our brain slices to ensure that the vast majority of synapses were statistically exposed to a SWCNT. We report that the presence of SWCNTs, for tens of minutes, do not alter the frequency of synaptic transmission in active hippocampal neuronal network (Figure S5B).

Based on this partition of the ECS environment definition (juxta- or non-juxta-synaptic), we next ran an extensive characterization of the ECS features pooled into these regions (Figure 3). In addition to information on local diffusivity, the analysis of SWCNT localizations also provides information on the local ECS dimensions applying an analytical approach described previously. (13) In short, SWCNT localizations were fitted to an ellipse over short periods of time (180 ms), where the shorter dimension represents the local ECS dimensions (ξ). Spatial maps of local diffusivities (Dinst/Dref) and ECS local dimensions were thus generated (Figure 3A), revealing that SWCNT diffusion is heterogeneous in all ECS areas. Due to the high neuronal density of brain tissues, we cannot exclude that the non-juxta-synaptic region may include synapses from other dendrites of non-infected (nonlabeled) neurons, so that non-juxta-synaptic behavior might be contaminated by juxta-synaptic features. In addition, because this work does not superlocalize SWCNT in 3D (nor synaptic centroids), the depth of focus of our microscope does not discriminate juxta-synaptic areas along the z (optical) axis of the microscope, such that juxta-synaptic regions might also be contaminated by non-juxta-synaptic features. In any case, we found that the juxta-synaptic diffusivity is 6-fold faster than the non-juxta-synaptic region (median_juxta = 0.089; median_non-juxta = 0.014; p < 0.001; Figure 3B) which might in fact represent a lower fold due to the two possible “contaminations” just mentioned.

Figure 3

Figure 3. Analysis of untreated organotypic brain slices. (A) Examples of individual trajectories. Localizations were divided based on the relative position to a GFP-PSD95 centroid (left panel: juxta-synaptic in red, non-juxta-synaptic in blue). Localizations farther than 1 μm are depicted in gray. Superlocalization analysis gave further information on local ECS dimensions (central panel) and diffusivity (right panel). Scale bars are 2 μm. (B) Cumulative distributions and violin plot and of diffusivity and (C) local dimensions. Graphs show that the juxta-synaptic nanoenvironment bears specific local properties (p < 0.001). (D) Scatterplots comparing juxta- and non-juxta-synaptic diffusivity and local dimensions of individual GFP-PSD95 positive clusters, showing that the diffusivity is higher and the local dimensions larger in the juxta area even at the single-synapse level.

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Figure 3C displays ECS dimension values in juxta- and non-juxta-synaptic domains. Similar to diffusivity, local ECS dimensions were highly heterogeneous, their widths ranging from around 50 nm (limited by the precision of our approach) to well above 1 μm. The vast majority (>70%) of local dimensions in the juxta-synaptic nanoenvironment were larger than 100 nm. Strikingly, the ECS local dimensions are significantly larger (∼2-fold) in the juxta-synaptic region as compared to the non-juxta-synaptic ones (median_juxta = 193 nm – median_non-juxta = 83 nm; p < 0.001; Figure 3C). Finally, we performed the comparison of diffusivity and local dimensions between juxta- and non-juxta-synaptic regions for each individual synaptic environment and represented each synapse on a scatter plot in Figure 3D. At the single synapse level, this analysis confirmed that higher diffusivity and larger local ECS dimensions are found in juxta-synaptic environments with respect to the local non-juxta-synaptic nanoenvironment. This observation has been confirmed by a matched paired analysis (p < 0.01).

In general, the broad shape of the cumulative distributions confirmed that the ECS is a highly heterogeneous milieu, where diverse local properties can impose a wide range of diffusivity and dimensional values. Indeed, in contrast to a free medium, diffusion in the brain ECS can be hindered by cell processes, astroglia, macromolecules of the matrix, wall drag, and the presence of charged molecules. In this environment, the diffusivity is also dependent on the hydrodynamic dimension of the diffusing probe, resulting in lower diffusivities for larger objects. Interestingly, median values of diffusivity and local dimensions evaluated at the level of individual juxta-synaptic regions are uncorrelated (Pearson’s r = 0.138; Figure S6A), suggesting that (i) the diffusivity of SWCNTs is mainly influenced by the molecular composition of the space, and (ii) spatial constrictions of cellular walls are not necessarily the central determinant of ECS diffusion inhomogeneities at the nanoscale near synapses.

As stated above, changes in neuronal activity are likely to alter ECS characteristics. (12,14,15) We thus now question whether these changes also alter ECS diffusivity and morphology in the juxta-synaptic nanoenvironment. We used two classical protocols to either favor (bicuculline, BIC 40 μM) or decrease (tetrodotoxin, TTX 2 μM) neuronal activity (Figure 4A). As expected, incubation with BIC significantly increased the basal activity, whereas TTX suppressed it (Figure 4B). Additionally, no significant differences were detected on the dimension of the GFP-PSD95 clusters (p = 0.1231, Figure S7).

Figure 4

Figure 4. Stimulation of organotypic brain slices. (A) Schematic of the stimulation. BIC or TTX treatments were applied for 24 h prior to SWCNT incubation to respectively block the inhibitory action of GABAA receptors or the sodium channels. (B) Representative electrophysiological traces of excitatory postsynaptic currents (EPSCs) from rat organotypic hippocampal slices DIV 12. Top traces recorded in the presence of aCSF only; bottom traces after addition of BIC or TTX in the medium to increase or decrease neuronal activity, respectively. (C) Examples of individual trajectories for BIC- and TTX-treated organotypic slices. As for control samples, localizations were divided based on the relative position to a GFP-PSD95 centroid (left panel: juxta-synaptic in red, extra-synaptic in blue). Localizations further than 1 μm are depicted in gray. Superlocalization analysis gave further information on local ECS dimensions (central panel) and diffusivity (right panel). Scale bars are 2 μm. (D) Cumulative distributions and violin plot of diffusivity for BIC-treated samples. Graphs show that BIC unified the synaptic microenvironment. (E) Cumulative distributions and violin plot of diffusivity for TTX-treated samples, showing a significant slowdown of diffusivity in the juxta-synaptic nanoenvironment. (F) Violin plot and cumulative distributions of local dimensions for BIC-treated samples, confirming the uniformity of the environment. (G) Violin plot and cumulative distributions of local dimensions for TTX-treated samples. For all graphs, the juxta-synaptic localizations are marked in red, while the non-juxta-synaptic ones are in blue. The gray boxes represent the localization precision of our analysis.

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Figure 4C shows examples of trajectories of SWCNTs in hippocampal tissues exposed to BIC or TTX. Similar to untreated samples, we partitioned nanotube localizations based on their juxta- or non-juxta-synaptic position. We observed that modulation of neuronal activity is accompanied by changes of the ECS local environment around synapses. More precisely, comparing ECS domains in each condition, we found that for BIC-treated samples the difference in diffusivity between regions near or distant from the GFP-PSD95 centroid became less pronounced (median_juxta_BIC = 0.032, median_non-juxta_BIC = 0.027, Figure 4D), whereas TTX yielded significantly lower values of diffusivity in the juxta-synaptic environment compared to non-juxta-synaptic spaces (median_juxta_TTX = 0.020, median_non-juxta_TTX = 0.038, Figure 4E). A similar alteration/modification was detected after the analysis of local dimensions in the juxta- and non-juxta-synaptic regions (median_juxta_BIC = 118 nm, median_non-juxta_BIC = 108 nm, Figure 4F; median_juxta_TTX = 112 nm, median_non-juxta_TTX = 174 nm, Figure 4G).

We next focus on the juxta-synaptic region to compare the different conditions (Figure 5). SWCNT diffusivity was slowed and ECS local dimensions were shrank in both BIC and TTX treatments when compared to control conditions (Figure 5A,B, p < 0.001). This observation suggests that the neuronal network can accommodate to neuronal activity changes (either increase or blockade) through ECS regulation. Finally, Figure 5 also indicates that in the juxta-synaptic region distributions of local diffusivity and dimensions of treated samples are less disperse than in control conditions, suggesting that BIC and TTX treatments standardized the juxta-synaptic environment.

Figure 5

Figure 5. Comparison of cumulative distributions of (A) local diffusivity and (B) local dimensions for control (gray), BIC-treated (green), and TTX-treated (purple) organotypic slices. When compared to control samples, both BIC and TTX slowed the SWCNT diffusivity in the juxta-synaptic nanoenvironment and narrowed down the local dimensions (p < 0.001). Local diffusivity in TTX-treated samples was significantly slower than in BIC-incubated tissues (p < 0.001), but the local dimensions remained comparable between the two conditions.

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A significant and global decrease in the ECS volume fraction and an increase in diffusion barriers have been reported during neuronal activity and pathological states. (23) These changes were related to cell swelling, cell loss, astrogliosis, rearrangement of neuronal and astrocytic processes, and changes in the extracellular matrix. Plastic changes in ECS volume, tortuosity, and anisotropy can also affect the communicaton between neurons and other cell types (e.g., astrocytes, oligodendrocytes). (5,6,24) Here, TTX-treated samples have slower diffusivity than the ones incubated with BIC (p < 0.001), but the local dimensions remain comparable between conditions, suggesting that the differences between BIC and TTX relies on the chemical modifications of the juxta-synaptic region. This is further supported by the correlation analysis evaluated for individual GFP-PSD95 positive clusters, which revealed a higher correlation between local diffusivity and dimensions in the juxta-synaptic region for BIC-treated with respect to TTX-treated samples (Figure S6B,C).

Altogether, our study revealed the existence of a juxta-synaptic ECS nanoenvironment within 500 nm from excitatory synapses in hippocampal brain slices, not accessible with previous approaches due to limited resolution. This observation was possible by correlating the dynamics and superlocalization of NIR-emitting carbon nanotube with HiLo microscopy of labeled synapses in live brain slices. Increasing or decreasing synaptic activity specifically modified the ECS diffusion and morphological parameters in the juxta-synaptic region. Such regulation of the ECS nanoenvironment around synapses would strongly influence the diffusion of neurotransmitters and modulators in the brain tissue, impacting neuronal network physiology and pathology.

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  • Corresponding Authors
  • Authors
    • Chiara Paviolo - Université de Bordeaux, Institut d’Optique & Centre National de la Recherche Scientifique, UMR 5298, 33400 Talence, France
    • Joana S. Ferreira - Université de Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, 33076 Bordeaux, France
    • Antony Lee - Université de Bordeaux, Institut d’Optique & Centre National de la Recherche Scientifique, UMR 5298, 33400 Talence, France;  Orcidhttps://orcid.org/0000-0003-2193-5369
    • Daniel Hunter - Université de Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, 33076 Bordeaux, France
    • Ivo Calaresu - Université de Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, 33076 Bordeaux, France
    • Somen Nandi - Université de Bordeaux, Institut d’Optique & Centre National de la Recherche Scientifique, UMR 5298, 33400 Talence, France
  • Author Contributions

    C.P. and J.S.F. contributed equally.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This work was performed with financial support from by the European Research Council Synergy grant (951294), Agence Nationale de la Recherche (ANR-15-CE16-0004-03), and the France-BioImaging National Infrastructure (ANR-10-INBS-04-01). C.P. acknowledges funding from EU’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant No 793296. A.L. acknowledges support from the Fondation ARC pour la recherche sur le cancer. S.N. acknowledges funding from EU’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant No 101024294. We thank the Bordeaux Imaging Center, a service unit of the CNRS-INSERM and Bordeaux University, the IINS Cell Biology Facility for organotypic culture slices preparation, in particular the help of Emeline Verdier, Delphine Bouchet Tessier and Constance Manso. We thank Christophe Mulle and Severine Deforges for providing the GFP-PSD95 lentivirus.

References

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    Transient changes in the size of the extracellular space in the sensorimotor cortex of cats in relation to stimulus-induced changes in potassium concentration
    Dietzel, I.; Heinemann, U.; Hofmeier, G.; Lux, H. D.
    Experimental Brain Research (1980), 40 (4), 432-9CODEN: EXBRAP; ISSN:0014-4819.
    The time course of local changes of the extracellular space (ES) was investigated by measuring concn. changes of repeatedly injected tetramethylammonium (TMA+) and choline (Ch+) ions for which cell membranes are largely impermeable. After stimulus-induced extracellular K+ concn. elevations, the changes in TMA+ and Ch+ concn. signals recorded with nominally K+-selective liq. ion-exchanger microelectrodes increased by ≤100%, thus indicating a redn. of the ES to 50% of its initial size. The shrinkage was maximal at sites where the K+ release into the ES was also largest. At very superficial and deep layers, however, considerable increases in extracellular K+ concn. were not accompanied by significant redns. in the ES. These findings can be explained as a consequence of K+ movement through spatially extended cell structures. Calcns. based on a model combining the spatial buffer mechanism to osmolarity changes caused by selective K+ transport through primarily K+-permeable membranes support this concept. Following stimulation addnl. iontophoretically induced extracellular K+ concn. rises were reduced in amplitude by ≤35%, even at sites where maximal decreases of the ES were obsd. This emphasizes the importance of active uptake for K+ clearance out of the ES.
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  5. 5
    Slais, K.; Vorisek, I.; Zoremba, N.; Homola, A.; Dmytrenko, L.; Sykova, E. Brain Metabolism and Diffusion in the Rat Cerebral Cortex during Pilocarpine-Induced Status Epilepticus. Exp. Neurol. 2008, 209 (1), 145– 154,  DOI: 10.1016/j.expneurol.2007.09.008
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    5
    Brain metabolism and diffusion in the rat cerebral cortex during pilocarpine-induced status epilepticus
    Slais, Karel; Vorisek, Ivan; Zoremba, Norbert; Homola, Ales; Dmytrenko, Lesia; Sykova, Eva
    Experimental Neurology (2008), 209 (1), 145-154CODEN: EXNEAC; ISSN:0014-4886. (Elsevier)
    The real-time iontophoretic method using tetramethylammonium-selective microelectrodes and diffusion-weighted magnetic resonance imaging were used to measure the extracellular space vol. fraction α, tortuosity λ and apparent diffusion coeff. of water (ADCW) 240 min after the administration of pilocarpine in urethane-anesthetized rats. The obtained data were correlated with extracellular lactate, glucose, and glutamate concns. and the lactate/pyruvate-ratio, detd. by intracerebral microdialysis. The control values of α and λ were 0.19 ± 0.004 and 1.58 ± 0.01, resp. Following pilocarpine application, α decreased to 0.134 ± 0.012 100 min later. Thereafter α increased, reaching 0.176 ± 0.009 140 min later. No significant changes in λ were obsd. during the entire time course of the expt. ADCW was significantly decreased 100 min after pilocarpine application (549 ± 8 μm2 s- 1) compared to controls (603 ± 11 μm2 s- 1); by the end of the expts., ADCW had returned to control values. The basal cortical levels of lactate, the lactate/pyruvate ratio, glucose and glutamate were 0.61 ± 0.05 mmol/l, 33.16 ± 4.26, 2.42 ± 0.13 mmol/l and 6.55 ± 1.31 μmol/l. Pilocarpine application led to a rise in lactate, the lactate/pyruvate ratio and glutamate levels, reaching 2.92 ± 0.60 mmol/l, 84.80 ± 11.72 and 22.39 ± 5.85 μmol/l within about 100 min, with a subsequent decrease to control values 140 min later. The time course of changes in glucose levels was different, with maximal levels of 3.49 ± 0.24 mmol/l reached 40 min after pilocarpine injection and a subsequent decrease to 1.25 ± 0.40 mmol/l obsd. 200 min later. Pathol. increased neuronal activity induced by pilocarpine causes cell swelling followed by a redn. in the ECS vol. fraction, which can contribute to the accumulation of toxic metabolites and lead to the start of epileptic discharges.
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  6. 6
    Colbourn, R.; Naik, A.; Hrabětová, S. ECS Dynamism and Its Influence on Neuronal Excitability and Seizures. Neurochem. Res. 2019, 44 (5), 1020– 1036,  DOI: 10.1007/s11064-019-02773-w
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    6
    ECS Dynamism and Its Influence on Neuronal Excitability and Seizures
    Colbourn, Robert; Naik, Aditi; Hrabetova, Sabina
    Neurochemical Research (2019), 44 (5), 1020-1036CODEN: NEREDZ; ISSN:0364-3190. (Springer)
    Seizure activity is governed by changes in normal neuronal physiol. that lead to a state of neuronal hyperexcitability and synchrony. There is a growing body of research and evidence suggesting that alterations in the vol. fraction (α) of the brain's extracellular space (ECS) have the ability to prolong or even initiate seizures. These ictogenic effects likely occur due to the ECS vol. being critically important in detg. both the concn. of neuroactive substances contained within it, such as ions and neurotransmitters, and the effect of elec. field-mediated interactions between neurons. Changes in the size of the ECS likely both precede a seizure, assisting in its initiation, and occur during a seizure, assisting in its maintenance. Different cellular ion and water transporters and channels are essential mediators in detg. neuronal excitability and synchrony and can do so through alterations in ECS vol. and/or through non-ECS vol. related mechanisms. This review will parse out the relationships between how the ECS vol. changes during normal physiol. and seizures, how those changes might alter neuronal physiol. to promote seizures, and what ion and water transporters and channels are important in linking ECS vol. changes and seizures.
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  7. 7
    Syková, E.; Nicholson, C. Diffusion in Brain Extracellular Space. Physiol. Rev. 2008, 88 (4), 1277– 1340,  DOI: 10.1152/physrev.00027.2007
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    7
    Diffusion in brain extracellular space
    Sykova, Eva; Nicholson, Charles
    Physiological Reviews (2008), 88 (4), 1277-1340CODEN: PHREA7; ISSN:0031-9333. (American Physiological Society)
    A review. Diffusion in the extracellular space (ECS) of the brain is constrained by the vol. fraction and the tortuosity and a modified diffusion equation represents the transport behavior of many mols. in the brain. Deviations from the equation reveal loss of mols. across the blood-brain barrier, through cellular uptake, binding, or other mechanisms. Early diffusion measurements used radiolabeled sucrose and other tracers. Presently, the real-time iontophoresis (RTI) method is employed for small ions and the integrative optical imaging (IOI) method for fluorescent macromols., including dextrans or proteins. Theor. models and simulations of the ECS have explored the influence of ECS geometry, effects of dead-space microdomains, extracellular matrix, and interaction of macromols. with ECS channels. Extensive exptl. studies with the RTI method employing the cation tetramethylammonium (TMA) in normal brain tissue show that the vol. fraction of the ECS typically is ∼20% and the tortuosity is ∼1.6 (i.e., free diffusion coeff. of TMA is reduced by 2.6), although there are regional variations. These parameters change during development and aging. Diffusion properties have been characterized in several interventions, including brain stimulation, osmotic challenge, and knockout of extracellular matrix components. Measurements have also been made during ischemia, in models of Alzheimer's and Parkinson's diseases, and in human gliomas. Overall, these studies improve our conception of ECS structure and the roles of glia and extracellular matrix in modulating the ECS microenvironment. Knowledge of ECS diffusion properties is valuable in contexts ranging from understanding extrasynaptic vol. transmission to the development of paradigms for drug delivery to the brain.
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  8. 8
    Zheng, K.; Jensen, T. P.; Savtchenko, L. P.; Levitt, J. A.; Suhling, K.; Rusakov, D. A. Nanoscale Diffusion in the Synaptic Cleft and beyond Measured with Time-Resolved Fluorescence Anisotropy Imaging. Sci. Rep. 2017, 7, 42022,  DOI: 10.1038/srep42022
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    8
    Nanoscale diffusion in the synaptic cleft and beyond measured with time-resolved fluorescence anisotropy imaging
    Zheng, Kaiyu; Jensen, Thomas P.; Savtchenko, Leonid P.; Levitt, James A.; Suhling, Klaus; Rusakov, Dmitri A.
    Scientific Reports (2017), 7 (), 42022CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
    Neural activity relies on mol. diffusion within nanoscopic spaces outside and inside nerve cells, such as synaptic clefts or dendritic spines. Measuring diffusion on this small scale in situ has not hitherto been possible, yet this knowledge is crit. for understanding the dynamics of mol. events and elec. currents that shape physiol. signals throughout the brain. Here we advance time-resolved fluorescence anisotropy imaging combined with two-photon excitation microscopy to map nanoscale diffusivity in ex vivo brain slices. We find that in the brain interstitial gaps small mols. move on av. ~ 30% slower than in a free medium whereas inside neuronal dendrites this retardation is ~ 70%. In the synaptic cleft free nanodiffusion is decelerated by ~ 46%. These quantities provide previously unattainable basic constrains for the receptor actions of released neurotransmitters, the elec. conductance of the brain interstitial space and the limiting rate of mol. interactions or conformational changes in the synaptic microenvironment.
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  9. 9
    Paviolo, C.; Cognet, L. Near-Infrared Nanoscopy with Carbon-Based Nanoparticles for the Exploration of the Brain Extracellular Space. Neurobiology of Disease 2021, 153, 105328,  DOI: 10.1016/j.nbd.2021.105328
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    9
    Near-infrared nanoscopy with carbon-based nanoparticles for the exploration of the brain extracellular space
    Paviolo, Chiara; Cognet, Laurent
    Neurobiology of Disease (2021), 153 (), 105328CODEN: NUDIEM; ISSN:0969-9961. (Elsevier Inc.)
    A review. Understanding the physiol. and pathol. of the brain requires detailed knowledge of its complex structures as well as dynamic internal processes at very different scales from the macro down to the mol. dimensions. A major yet poorly described brain compartment is the brain extracellular space (ECS). Signalling mols. rapidly diffuse through the brain ECS which is complex and dynamic structure at numerous lengths and time scales. In recent years, characterization of the ECS using nanomaterials has made remarkable progress, including local anal. of nanoscopic dimensions and diffusivity as well as local chem. sensing. In particular, carbon nanomaterials combined with advanced optical technologies, biochem. and biophys. anal., offer novel promises for understanding the ECS morphol. as well as neuron connectivity and neurochem. In this review, we present the state-of-the-art in this quest, which mainly focuses on a type of carbon nanomaterial, single walled carbon nanotubes, as fluorescent nanoprobes to unveil the ECS features in the nanometer domain.
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  10. 10
    Welsher, K.; Liu, Z.; Sherlock, S. P.; Robinson, J. T.; Chen, Z.; Daranciang, D.; Dai, H. A Route to Brightly Fluorescent Carbon Nanotubes for Near-Infrared Imaging in Mice. Nat. Nanotechnol. 2009, 4 (11), 773– 780,  DOI: 10.1038/nnano.2009.294
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    10
    A route to brightly fluorescent carbon nanotubes for near-infrared imaging in mice
    Welsher, Kevin; Liu, Zhuang; Sherlock, Sarah P.; Robinson, Joshua Tucker; Chen, Zhuo; Daranciang, Dan; Dai, Hongjie
    Nature Nanotechnology (2009), 4 (11), 773-780CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
    The near-IR photoluminescence intrinsic to semiconducting single-walled carbon nanotubes is ideal for biol. imaging owing to the low autofluorescence and deep tissue penetration in the near-IR region beyond 1 μm. However, biocompatible single-walled carbon nanotubes with high quantum yield have been elusive. Here, we show that sonicating single-walled carbon nanotubes with sodium cholate, followed by surfactant exchange to form phospholipid-polyethylene glycol coated nanotubes, produces in vivo imaging agents that are both bright and biocompatible. The exchange procedure is better than directly sonicating the tubes with the phospholipid-polyethylene glycol, because it results in less damage to the nanotubes and improves the quantum yield. We show whole-animal in vivo imaging using an InGaAs camera in the 1-1.7 μm spectral range by detecting the intrinsic near-IR photoluminescence of the exchange' single-walled carbon nanotubes at a low dose (17 mg l-1 injected dose). The deep tissue penetration and low autofluorescence background allowed high-resoln. intravital microscopy imaging of tumor vessels beneath thick skin.
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  11. 11
    Kruss, S.; Landry, M. P.; Vander Ende, E.; Lima, B. M. A.; Reuel, N. F.; Zhang, J.; Nelson, J.; Mu, B.; Hilmer, A.; Strano, M. Neurotransmitter Detection Using Corona Phase Molecular Recognition on Fluorescent Single-Walled Carbon Nanotube Sensors. J. Am. Chem. Soc. 2014, 136 (2), 713– 724,  DOI: 10.1021/ja410433b
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    11
    Neurotransmitter Detection Using Corona Phase Molecular Recognition on Fluorescent Single-Walled Carbon Nanotube Sensors
    Kruss, Sebastian; Landry, Markita P.; Vander Ende, Emma; Lima, Barbara M. A.; Reuel, Nigel F.; Zhang, Jingqing; Nelson, Justin; Mu, Bin; Hilmer, Andrew; Strano, Michael
    Journal of the American Chemical Society (2014), 136 (2), 713-724CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    Temporal and spatial changes in neurotransmitter concns. are central to information processing in neural networks. Therefore, biosensors for neurotransmitters are essential tools for neuroscience. The authors applied a new technique, corona phase mol. recognition (CoPhMoRe), to identify adsorbed polymer phases on fluorescent single-walled carbon nanotubes (SWCNTs) that allow for the selective detection of specific neurotransmitters, including dopamine. The authors functionalized and suspended SWCNTs with a library of different polymers (n = 30) contg. phospholipids, nucleic acids, and amphiphilic polymers to study how neurotransmitters modulate the resulting band gap, near-IR (nIR) fluorescence of the SWCNT. The authors identified several corona phases that enable the selective detection of neurotransmitters. Catecholamines such as dopamine increased the fluorescence of specific single-stranded DNA- and RNA-wrapped SWCNTs by 58-80% upon addn. of 100 μM dopamine depending on the SWCNT chirality (n,m). In soln., the limit of detection was 11 nM [Kd = 433 nM for (GT)15 DNA-wrapped SWCNTs]. Mechanistic studies revealed that this turn-on response is due to an increase in fluorescence quantum yield and not covalent modification of the SWCNT or scavenging of reactive oxygen species. When immobilized on a surface, the fluorescence intensity of a single DNA- or RNA-wrapped SWCNT is enhanced by a factor of up to 5.39±1.44, whereby fluorescence signals are reversible. The authors' findings indicate that certain DNA/RNA coronae act as conformational switches on SWCNTs, which reversibly modulate the SWCNT fluorescence. These findings suggest that the authors' polymer-SWCNT constructs can act as fluorescent neurotransmitter sensors in the tissue-compatible nIR optical window, which may find applications in neuroscience.
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  12. 12
    Godin, A. G.; Varela, J. A.; Gao, Z.; Danné, N.; Dupuis, J. P.; Lounis, B.; Groc, L.; Cognet, L. Single-Nanotube Tracking Reveals the Nanoscale Organization of the Extracellular Space in the Live Brain. Nat. Nanotechnol. 2017, 12 (3), 238– 243,  DOI: 10.1038/nnano.2016.248
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    12
    Single-nanotube tracking reveals the nanoscale organization of the extracellular space in the live brain
    Godin, Antoine G.; Varela, Juan A.; Gao, Zhenghong; Danne, Noemie; Dupuis, Julien P.; Lounis, Brahim; Groc, Laurent; Cognet, Laurent
    Nature Nanotechnology (2017), 12 (3), 238-243CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
    The brain is a dynamic structure with the extracellular space (ECS) taking up almost a quarter of its vol. Signaling mols., neurotransmitters and nutrients transit via the ECS, which constitutes a key microenvironment for cellular communication and the clearance of toxic metabolites. The spatial organization of the ECS varies during sleep, development and aging and is probably altered in neuropsychiatric and degenerative diseases, as inferred from electron microscopy and macroscopic biophys. studies. Here the authors show an approach to directly observe the local ECS structures and rheol. in brain tissue using super-resoln. imaging. The authors inject single-walled carbon nanotubes into rat cerebroventricles and follow the near-IR emission of individual nanotubes as they diffuse inside the ECS for tens of minutes in acute slices. Because of the interplay between the nanotube geometry and the ECS local environment, the authors can ext. information about the dimensions and local viscosity of the ECS. A striking diversity of ECS dimensions down to 40 nm and of local viscosity values were found. Moreover, by chem. altering the extracellular matrix of the brains of live animals before nanotube injection, the authors reveal that the rheol. properties of the ECS are affected, but these alterations are local and inhomogeneous at the nanoscale.
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  13. 13
    Paviolo, C.; Soria, F. N.; Ferreira, J. S.; Lee, A.; Groc, L.; Bezard, E.; Cognet, L. Nanoscale Exploration of the Extracellular Space in the Live Brain by Combining Single Carbon Nanotube Tracking and Super-Resolution Imaging Analysis. Methods 2020, 174, 91– 99,  DOI: 10.1016/j.ymeth.2019.03.005
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    13
    Nanoscale exploration of the extracellular space in the live brain by combining single carbon nanotube tracking and super-resolution imaging analysis
    Paviolo, Chiara; Soria, Federico N.; Ferreira, Joana S.; Lee, Antony; Groc, Laurent; Bezard, Erwan; Cognet, Laurent
    Methods (Amsterdam, Netherlands) (2020), 174 (), 91-99CODEN: MTHDE9; ISSN:1046-2023. (Elsevier B.V.)
    The brain extracellular space (ECS) is a system of narrow compartments whose intricate nanometric structure has remained elusive until very recently. Understanding such a complex organization represents a technol. challenge that requires a technique able to resolve these nanoscopic spaces and simultaneously characterize their rheol. properties. We recently used single-walled carbon nanotubes (SWCNTs) as near-IR fluorescent probes to map with nanoscale precision the local organization and rheol. of the ECS. Here we expand our method by tracking single nanotubes through super-resoln. imaging in rat organotypic hippocampal slices and acute brain slices from adult mice, pioneering the exploration of the adult brain ECS at the nanoscale. We found a highly heterogeneous ECS, where local rheol. properties can change drastically within few nanometers. Our results suggest differences in local ECS diffusion environments in organotypic slices when compared to adult mouse slices. Data obtained from super-resolved maps of the SWCNT trajectories indicate that ECS widths may vary between brain tissue models, with a looser, less crowded nano-environment in organotypic cultured slices.
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  14. 14
    Tønnesen, J.; Inavalli, V. V. G. K.; Nägerl, U. V. Super-Resolution Imaging of the Extracellular Space in Living Brain Tissue. Cell 2018, 172 (5), 1108– 1121,  DOI: 10.1016/j.cell.2018.02.007
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    14
    Super-Resolution Imaging of the Extracellular Space in Living Brain Tissue
    Tonnesen Jan; Inavalli V V G Krishna; Nagerl U Valentin
    Cell (2018), 172 (5), 1108-1121.e15 ISSN:.
    The extracellular space (ECS) of the brain has an extremely complex spatial organization, which has defied conventional light microscopy. Consequently, despite a marked interest in the physiological roles of brain ECS, its structure and dynamics remain largely inaccessible for experimenters. We combined 3D-STED microscopy and fluorescent labeling of the extracellular fluid to develop super-resolution shadow imaging (SUSHI) of brain ECS in living organotypic brain slices. SUSHI enables quantitative analysis of ECS structure and reveals dynamics on multiple scales in response to a variety of physiological stimuli. Because SUSHI produces sharp negative images of all cellular structures, it also enables unbiased imaging of unlabeled brain cells with respect to their anatomical context. Moreover, the extracellular labeling strategy greatly alleviates problems of photobleaching and phototoxicity associated with traditional imaging approaches. As a straightforward variant of STED microscopy, SUSHI provides unprecedented access to the structure and dynamics of live brain ECS and neuropil.
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  15. 15
    Soria, F. N.; Paviolo, C.; Doudnikoff, E.; Arotcarena, M.-L.; Lee, A.; Danné, N.; Mandal, A. K.; Gosset, P.; Dehay, B.; Groc, L.; Cognet, L.; Bezard, E. Synucleinopathy Alters Nanoscale Organization and Diffusion in the Brain Extracellular Space through Hyaluronan Remodeling. Nat. Commun. 2020, 11 (1), 3440,  DOI: 10.1038/s41467-020-17328-9
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    15
    Synucleinopathy alters nanoscale organization and diffusion in the brain extracellular space through hyaluronan remodeling
    Soria, Federico N.; Paviolo, Chiara; Doudnikoff, Evelyne; Arotcarena, Marie-Laure; Lee, Antony; Danne, Noemie; Mandal, Amit Kumar; Gosset, Philippe; Dehay, Benjamin; Groc, Laurent; Cognet, Laurent; Bezard, Erwan
    Nature Communications (2020), 11 (1), 3440CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    Abstr.: In recent years, exploration of the brain extracellular space (ECS) has made remarkable progress, including nanoscopic characterizations. However, whether ECS precise conformation is altered during brain pathol. remains unknown. Here we study the nanoscale organization of pathol. ECS in adult mice under degenerative conditions. Using electron microscopy in cryofixed tissue and single nanotube tracking in live brain slices combined with super-resoln. imaging anal., we find enlarged ECS dimensions and increased nanoscale diffusion after α-synuclein-induced neurodegeneration. These animals display a degraded hyaluronan matrix in areas close to reactive microglia. Furthermore, exptl. hyaluronan depletion in vivo reduces dopaminergic cell loss and α-synuclein load, induces microgliosis and increases ECS diffusivity, highlighting hyaluronan as diffusional barrier and local tissue organizer. These findings demonstrate the interplay of ECS, extracellular matrix and glia in pathol., unraveling ECS features relevant for the α-synuclein propagation hypothesis and suggesting matrix manipulation as a disease-modifying strategy.
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  16. 16
    Lim, D.; Ford, T. N.; Chu, K. K.; Mertz, J. Optically Sectioned in Vivo Imaging with Speckle Illumination HiLo Microscopy. J. Biomed. Opt. 2011, 16 (1), 016014– 016014–016018,  DOI: 10.1117/1.3528656
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    16
    Optically sectioned in vivo imaging with speckle illumination HiLo microscopy
    Lim Daryl; Ford Tim N; Chu Kengyeh K; Mertz Jerome
    Journal of biomedical optics (2011), 16 (1), 016014 ISSN:.
    We present a simple wide-field imaging technique, called HiLo microscopy, that is capable of producing optically sectioned images in real time, comparable in quality to confocal laser scanning microscopy. The technique is based on the fusion of two raw images, one acquired with speckle illumination and another with standard uniform illumination. The fusion can be numerically adjusted, using a single parameter, to produce optically sectioned images of varying thicknesses with the same raw data. Direct comparison between our HiLo microscope and a commercial confocal laser scanning microscope is made on the basis of sectioning strength and imaging performance. Specifically, we show that HiLo and confocal 3-D imaging of a GFP-labeled mouse brain hippocampus are comparable in quality. Moreover, HiLo microscopy is capable of faster, near video rate imaging over larger fields of view than attainable with standard confocal microscopes. The goal of this paper is to advertise the simplicity, robustness, and versatility of HiLo microscopy, which we highlight with in vivo imaging of common model organisms including planaria, C. elegans, and zebrafish.
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  17. 17
    Lim, D.; Chu, K. K.; Mertz, J. Wide-Field Fluorescence Sectioning with Hybrid Speckle and Uniform-Illumination Microscopy. Opt. Lett. 2008, 33 (16), 1819– 1821,  DOI: 10.1364/OL.33.001819
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    17
    Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy
    Lim Daryl; Chu Kengyeh K; Mertz Jerome
    Optics letters (2008), 33 (16), 1819-21 ISSN:0146-9592.
    We describe a method of obtaining optical sectioning with a standard wide-field fluorescence microscope. The method involves acquiring two images, one with nonuniform illumination (in our case, speckle) and another with uniform illumination (in our case, randomized speckle). An evaluation of the local contrast in the speckle-illumination image provides an optically sectioned image with low resolution. This is complemented with high-resolution information obtained from the uniform-illumination image. A fusion of both images leads to a full resolution image that is optically sectioned across all spatial frequencies. This hybrid illumination method is fast, robust, and generalizable to a variety of illumination and imaging configurations.
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  18. 18
    Gao, Z.; Danné, N.; Godin, A. G.; Lounis, B.; Cognet, L. Evaluation of Different Single-Walled Carbon Nanotube Surface Coatings for Single-Particle Tracking Applications in Biological Environments. Nanomaterials 2017, 7 (11), 393,  DOI: 10.3390/nano7110393
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    18
    Evaluation of different single-walled carbon nanotube surface coatings for single-particle tracking applications in biological environments
    Gao, Zhenghong; Danne, Noemie; Godin, Antoine Guillaume; Lounis, Brahim; Cognet, Laurent
    Nanomaterials (2017), 7 (11), 393/1-393/12CODEN: NANOKO; ISSN:2079-4991. (MDPI AG)
    Fluorescence imaging of biol. systems down to the single-mol. level has generated many advances in cellular biol. For applications within intact tissue, single-walled carbon nanotubes (SWCNTs) are emerging as distinctive single-mol. nanoprobes, due to their near-IR photoluminescence properties. For this, SWCNT surfaces must be coated using adequate mol. moieties. Yet, the choice of the suspension agent is crit. since it influences both the chem. and emission properties of the SWCNTs within their environment. Here, we compare the most commonly used surface coatings for encapsulating photoluminescent SWCNTs in the context of bio-imaging applications. To be applied as single-mol. nanoprobes, encapsulated nanotubes should display low cytotoxicity, and minimal unspecific interactions with cells while still being highly luminescent so as to be imaged and tracked down to the single nanotube level for long periods of time. We tested the cell proliferation and cellular viability of each surface coating and evaluated the impact of the biocompatible surface coatings on nanotube photoluminescence brightness. Our study establishes that phospholipid-polyethylene glycol-coated carbon nanotube is the best current choice for single nanotube tracking expts. in live biol. samples.
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    ACS Photonics (2018), 5 (2), 359-364CODEN: APCHD5; ISSN:2330-4022. (American Chemical Society)
    Luminescent single-walled carbon nanotubes (SWCNTs) are unique nanoemitters that allow near-IR single-mol. detection within biol. tissues. Interestingly, the recent discovery of upconversion luminescence from (6,5) SWCNTs provides a novel opportunity for deep tissue single SWCNT detection. Yet, the optimal excitation strategy for video-rate imaging of individual SWCNTs within live tissues needs to be detd. taking into account the constraints imposed by the biol. matter. Here, the authors directly compare the luminescence efficiencies of single (6,5) SWCNTs excited by continuous-wave lasers at their second-order excitonic transition, at their K-momentum exciton-phonon sideband, or through upconversion. For these three excitations spanning visible to near-IR wavelengths, the relevance of single SWCNT imaging is considered inside brain tissue. The effects of tissue scattering, absorption, autofluorescence, and temp. increase induced by excitation light are systematically examd.
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    Brownian Motion of Stiff Filaments in a Crowded Environment
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    The thermal motion of stiff filaments in a crowded environment is highly constrained and anisotropic; it underlies the behavior of such disparate systems as polymer materials, nanocomposites, and the cell cytoskeleton. Despite decades of theor. study, the fundamental dynamics of such systems remains a mystery. Using near-IR video microscopy, we studied the thermal diffusion of individual single-walled carbon nanotubes (SWNTs) confined in porous agarose networks. We found that even a small bending flexibility of SWNTs strongly enhances their motion: The rotational diffusion const. is proportional to the filament-bending compliance and is independent of the network pore size. The interplay between crowding and thermal bending implies that the notion of a filament's stiffness depends on its confinement. Moreover, the mobility of SWNTs and other inclusions can be controlled by tailoring their stiffness.
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    Philosophical transactions of the Royal Society of London. Series B, Biological sciences (2014), 369 (1654), 20130608 ISSN:.
    Volume transmission is a form of intercellular communication that does not require synapses; it is based on the diffusion of neuroactive substances across the brain extracellular space (ECS) and their binding to extrasynaptic high-affinity receptors on neurons or glia. Extracellular diffusion is restricted by the limited volume of the ECS, which is described by the ECS volume fraction α, and the presence of diffusion barriers, reflected by tortuosity λ, that are created, for example, by fine astrocytic processes or extracellular matrix (ECM) molecules. Organized astrocytic processes, ECM scaffolds or myelin sheets channel the extracellular diffusion so that it is facilitated in a certain direction, i.e. anisotropic. The diffusion properties of the ECS are profoundly influenced by various processes such as the swelling and morphological rebuilding of astrocytes during either transient or persisting physiological or pathological states, or the remodelling of the ECM in tumorous or epileptogenic tissue, during Alzheimer's disease, after enzymatic treatment or in transgenic animals. The changing diffusion properties of the ECM influence neuron-glia interaction, learning abilities, the extent of neuronal damage and even cell migration. From a clinical point of view, diffusion parameter changes occurring during pathological states could be important for diagnosis, drug delivery and treatment.
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    Proceedings of the National Academy of Sciences of the United States of America (2004), 101 (7), 2151-2155CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Interactions between sep. synaptic inputs converging on the same target appear to contribute to the fine-tuning of information processing in the central nervous system. Intersynaptic crosstalk is made possible by transmitter spillover from the synaptic cleft and its diffusion over a distance to neighboring synapses. This is the case for glutamate, which inhibits γ-aminobutyric acid (GABA)ergic transmission in several brain regions through the activation of presynaptic receptors. Such heterosynaptic modulation depends on factors that influence diffusion in the extracellular space (ECS). Because glial cells represent a phys. barrier to diffusion and, in addn., are essential for glutamate uptake, the authors investigated the physiol. contribution of the astrocytic environment of neurons to glutamate-mediated intersynaptic communication in the brain. The reduced astrocytic coverage of magnocellular neurons occurring in the supraoptic nucleus of lactating rats facilitates diffusion in the ECS, as revealed by tortuosity and vol. fraction measurements. Under these conditions, glutamate spillover, monitored through metabotropic glutamate receptor-mediated depression of GABAergic transmission, is greatly enhanced. Conversely, impeding diffusion with dextran largely prevents crosstalk between glutamatergic and GABAergic afferent inputs. Astrocytes, therefore, by hindering diffusion in the ECS, regulate intersynaptic communication between neighboring synapses and, probably, overall vol. transmission in the brain.
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  • Abstract

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    Figure 1

    Figure 1. Experimental conditions. (A) Schematic of the experimental setup. Imaging was performed on a customized epifluorescent microscope equipped with a speckle based illumination setup. NIR imaging of SWCNTs was performed at 845 nm with a 60× immersion objective. GFP-PSD95 expressing neurons were imaged at 488 nm through the same objective and recorded by a HiLo modality. Individual SWCNTs were followed in the ECS of living brains using an EM-CCD camera. A standard white light fiber and low magnification objective (4×) were initially used to check the CA1 position in the hippocampal slice. (B) Schematic of the organotypic slices. Rat organotypic cultures were prepared from rat pup brains and cultured for 4 days in vitro (DIV) before PSD95 infection. SWCNTs were incubated at DIV 12–14 for 2 h. (C) Example of a speckle and HiLo images recorded with the Sparq system. Individual GFP-PSD95 clusters were clearly recognizable. Scale bars are 20 μm. (D) Typical recording of SWCNTs in living organotypic slices. Nanotubes were sparsely and individually detected at high signal-to-noise ratio. Scale bar is 20 μm.

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    Figure 2

    Figure 2. (A) Schematic of the analysis. Individual SWCNT trajectories were analyzed on coronal areas of 100 nm from the synaptic centroids. Scale bar is 1 μm. (B) Median (red crosses), 25th/75th percentile (shaded area), and cumulative distribution functions (inset) of the diffusivity values calculated at each coronal area for control conditions (31 SWCNTs). A sharp transition in the diffusivity values was observed between 400 and 500 nm from the PSD95 centroids.

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    Figure 3

    Figure 3. Analysis of untreated organotypic brain slices. (A) Examples of individual trajectories. Localizations were divided based on the relative position to a GFP-PSD95 centroid (left panel: juxta-synaptic in red, non-juxta-synaptic in blue). Localizations farther than 1 μm are depicted in gray. Superlocalization analysis gave further information on local ECS dimensions (central panel) and diffusivity (right panel). Scale bars are 2 μm. (B) Cumulative distributions and violin plot and of diffusivity and (C) local dimensions. Graphs show that the juxta-synaptic nanoenvironment bears specific local properties (p < 0.001). (D) Scatterplots comparing juxta- and non-juxta-synaptic diffusivity and local dimensions of individual GFP-PSD95 positive clusters, showing that the diffusivity is higher and the local dimensions larger in the juxta area even at the single-synapse level.

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    Figure 4

    Figure 4. Stimulation of organotypic brain slices. (A) Schematic of the stimulation. BIC or TTX treatments were applied for 24 h prior to SWCNT incubation to respectively block the inhibitory action of GABAA receptors or the sodium channels. (B) Representative electrophysiological traces of excitatory postsynaptic currents (EPSCs) from rat organotypic hippocampal slices DIV 12. Top traces recorded in the presence of aCSF only; bottom traces after addition of BIC or TTX in the medium to increase or decrease neuronal activity, respectively. (C) Examples of individual trajectories for BIC- and TTX-treated organotypic slices. As for control samples, localizations were divided based on the relative position to a GFP-PSD95 centroid (left panel: juxta-synaptic in red, extra-synaptic in blue). Localizations further than 1 μm are depicted in gray. Superlocalization analysis gave further information on local ECS dimensions (central panel) and diffusivity (right panel). Scale bars are 2 μm. (D) Cumulative distributions and violin plot of diffusivity for BIC-treated samples. Graphs show that BIC unified the synaptic microenvironment. (E) Cumulative distributions and violin plot of diffusivity for TTX-treated samples, showing a significant slowdown of diffusivity in the juxta-synaptic nanoenvironment. (F) Violin plot and cumulative distributions of local dimensions for BIC-treated samples, confirming the uniformity of the environment. (G) Violin plot and cumulative distributions of local dimensions for TTX-treated samples. For all graphs, the juxta-synaptic localizations are marked in red, while the non-juxta-synaptic ones are in blue. The gray boxes represent the localization precision of our analysis.

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    Figure 5

    Figure 5. Comparison of cumulative distributions of (A) local diffusivity and (B) local dimensions for control (gray), BIC-treated (green), and TTX-treated (purple) organotypic slices. When compared to control samples, both BIC and TTX slowed the SWCNT diffusivity in the juxta-synaptic nanoenvironment and narrowed down the local dimensions (p < 0.001). Local diffusivity in TTX-treated samples was significantly slower than in BIC-incubated tissues (p < 0.001), but the local dimensions remained comparable between the two conditions.

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      Dietzel, I.; Heinemann, U.; Hofmeier, G.; Lux, H. D.
      Experimental Brain Research (1980), 40 (4), 432-9CODEN: EXBRAP; ISSN:0014-4819.
      The time course of local changes of the extracellular space (ES) was investigated by measuring concn. changes of repeatedly injected tetramethylammonium (TMA+) and choline (Ch+) ions for which cell membranes are largely impermeable. After stimulus-induced extracellular K+ concn. elevations, the changes in TMA+ and Ch+ concn. signals recorded with nominally K+-selective liq. ion-exchanger microelectrodes increased by ≤100%, thus indicating a redn. of the ES to 50% of its initial size. The shrinkage was maximal at sites where the K+ release into the ES was also largest. At very superficial and deep layers, however, considerable increases in extracellular K+ concn. were not accompanied by significant redns. in the ES. These findings can be explained as a consequence of K+ movement through spatially extended cell structures. Calcns. based on a model combining the spatial buffer mechanism to osmolarity changes caused by selective K+ transport through primarily K+-permeable membranes support this concept. Following stimulation addnl. iontophoretically induced extracellular K+ concn. rises were reduced in amplitude by ≤35%, even at sites where maximal decreases of the ES were obsd. This emphasizes the importance of active uptake for K+ clearance out of the ES.
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    5. 5
      Slais, K.; Vorisek, I.; Zoremba, N.; Homola, A.; Dmytrenko, L.; Sykova, E. Brain Metabolism and Diffusion in the Rat Cerebral Cortex during Pilocarpine-Induced Status Epilepticus. Exp. Neurol. 2008, 209 (1), 145– 154,  DOI: 10.1016/j.expneurol.2007.09.008
      5
      Brain metabolism and diffusion in the rat cerebral cortex during pilocarpine-induced status epilepticus
      Slais, Karel; Vorisek, Ivan; Zoremba, Norbert; Homola, Ales; Dmytrenko, Lesia; Sykova, Eva
      Experimental Neurology (2008), 209 (1), 145-154CODEN: EXNEAC; ISSN:0014-4886. (Elsevier)
      The real-time iontophoretic method using tetramethylammonium-selective microelectrodes and diffusion-weighted magnetic resonance imaging were used to measure the extracellular space vol. fraction α, tortuosity λ and apparent diffusion coeff. of water (ADCW) 240 min after the administration of pilocarpine in urethane-anesthetized rats. The obtained data were correlated with extracellular lactate, glucose, and glutamate concns. and the lactate/pyruvate-ratio, detd. by intracerebral microdialysis. The control values of α and λ were 0.19 ± 0.004 and 1.58 ± 0.01, resp. Following pilocarpine application, α decreased to 0.134 ± 0.012 100 min later. Thereafter α increased, reaching 0.176 ± 0.009 140 min later. No significant changes in λ were obsd. during the entire time course of the expt. ADCW was significantly decreased 100 min after pilocarpine application (549 ± 8 μm2 s- 1) compared to controls (603 ± 11 μm2 s- 1); by the end of the expts., ADCW had returned to control values. The basal cortical levels of lactate, the lactate/pyruvate ratio, glucose and glutamate were 0.61 ± 0.05 mmol/l, 33.16 ± 4.26, 2.42 ± 0.13 mmol/l and 6.55 ± 1.31 μmol/l. Pilocarpine application led to a rise in lactate, the lactate/pyruvate ratio and glutamate levels, reaching 2.92 ± 0.60 mmol/l, 84.80 ± 11.72 and 22.39 ± 5.85 μmol/l within about 100 min, with a subsequent decrease to control values 140 min later. The time course of changes in glucose levels was different, with maximal levels of 3.49 ± 0.24 mmol/l reached 40 min after pilocarpine injection and a subsequent decrease to 1.25 ± 0.40 mmol/l obsd. 200 min later. Pathol. increased neuronal activity induced by pilocarpine causes cell swelling followed by a redn. in the ECS vol. fraction, which can contribute to the accumulation of toxic metabolites and lead to the start of epileptic discharges.
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    6. 6
      Colbourn, R.; Naik, A.; Hrabětová, S. ECS Dynamism and Its Influence on Neuronal Excitability and Seizures. Neurochem. Res. 2019, 44 (5), 1020– 1036,  DOI: 10.1007/s11064-019-02773-w
      6
      ECS Dynamism and Its Influence on Neuronal Excitability and Seizures
      Colbourn, Robert; Naik, Aditi; Hrabetova, Sabina
      Neurochemical Research (2019), 44 (5), 1020-1036CODEN: NEREDZ; ISSN:0364-3190. (Springer)
      Seizure activity is governed by changes in normal neuronal physiol. that lead to a state of neuronal hyperexcitability and synchrony. There is a growing body of research and evidence suggesting that alterations in the vol. fraction (α) of the brain's extracellular space (ECS) have the ability to prolong or even initiate seizures. These ictogenic effects likely occur due to the ECS vol. being critically important in detg. both the concn. of neuroactive substances contained within it, such as ions and neurotransmitters, and the effect of elec. field-mediated interactions between neurons. Changes in the size of the ECS likely both precede a seizure, assisting in its initiation, and occur during a seizure, assisting in its maintenance. Different cellular ion and water transporters and channels are essential mediators in detg. neuronal excitability and synchrony and can do so through alterations in ECS vol. and/or through non-ECS vol. related mechanisms. This review will parse out the relationships between how the ECS vol. changes during normal physiol. and seizures, how those changes might alter neuronal physiol. to promote seizures, and what ion and water transporters and channels are important in linking ECS vol. changes and seizures.
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    7. 7
      Syková, E.; Nicholson, C. Diffusion in Brain Extracellular Space. Physiol. Rev. 2008, 88 (4), 1277– 1340,  DOI: 10.1152/physrev.00027.2007
      7
      Diffusion in brain extracellular space
      Sykova, Eva; Nicholson, Charles
      Physiological Reviews (2008), 88 (4), 1277-1340CODEN: PHREA7; ISSN:0031-9333. (American Physiological Society)
      A review. Diffusion in the extracellular space (ECS) of the brain is constrained by the vol. fraction and the tortuosity and a modified diffusion equation represents the transport behavior of many mols. in the brain. Deviations from the equation reveal loss of mols. across the blood-brain barrier, through cellular uptake, binding, or other mechanisms. Early diffusion measurements used radiolabeled sucrose and other tracers. Presently, the real-time iontophoresis (RTI) method is employed for small ions and the integrative optical imaging (IOI) method for fluorescent macromols., including dextrans or proteins. Theor. models and simulations of the ECS have explored the influence of ECS geometry, effects of dead-space microdomains, extracellular matrix, and interaction of macromols. with ECS channels. Extensive exptl. studies with the RTI method employing the cation tetramethylammonium (TMA) in normal brain tissue show that the vol. fraction of the ECS typically is ∼20% and the tortuosity is ∼1.6 (i.e., free diffusion coeff. of TMA is reduced by 2.6), although there are regional variations. These parameters change during development and aging. Diffusion properties have been characterized in several interventions, including brain stimulation, osmotic challenge, and knockout of extracellular matrix components. Measurements have also been made during ischemia, in models of Alzheimer's and Parkinson's diseases, and in human gliomas. Overall, these studies improve our conception of ECS structure and the roles of glia and extracellular matrix in modulating the ECS microenvironment. Knowledge of ECS diffusion properties is valuable in contexts ranging from understanding extrasynaptic vol. transmission to the development of paradigms for drug delivery to the brain.
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    8. 8
      Zheng, K.; Jensen, T. P.; Savtchenko, L. P.; Levitt, J. A.; Suhling, K.; Rusakov, D. A. Nanoscale Diffusion in the Synaptic Cleft and beyond Measured with Time-Resolved Fluorescence Anisotropy Imaging. Sci. Rep. 2017, 7, 42022,  DOI: 10.1038/srep42022
      8
      Nanoscale diffusion in the synaptic cleft and beyond measured with time-resolved fluorescence anisotropy imaging
      Zheng, Kaiyu; Jensen, Thomas P.; Savtchenko, Leonid P.; Levitt, James A.; Suhling, Klaus; Rusakov, Dmitri A.
      Scientific Reports (2017), 7 (), 42022CODEN: SRCEC3; ISSN:2045-2322. (Nature Publishing Group)
      Neural activity relies on mol. diffusion within nanoscopic spaces outside and inside nerve cells, such as synaptic clefts or dendritic spines. Measuring diffusion on this small scale in situ has not hitherto been possible, yet this knowledge is crit. for understanding the dynamics of mol. events and elec. currents that shape physiol. signals throughout the brain. Here we advance time-resolved fluorescence anisotropy imaging combined with two-photon excitation microscopy to map nanoscale diffusivity in ex vivo brain slices. We find that in the brain interstitial gaps small mols. move on av. ~ 30% slower than in a free medium whereas inside neuronal dendrites this retardation is ~ 70%. In the synaptic cleft free nanodiffusion is decelerated by ~ 46%. These quantities provide previously unattainable basic constrains for the receptor actions of released neurotransmitters, the elec. conductance of the brain interstitial space and the limiting rate of mol. interactions or conformational changes in the synaptic microenvironment.
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    9. 9
      Paviolo, C.; Cognet, L. Near-Infrared Nanoscopy with Carbon-Based Nanoparticles for the Exploration of the Brain Extracellular Space. Neurobiology of Disease 2021, 153, 105328,  DOI: 10.1016/j.nbd.2021.105328
      9
      Near-infrared nanoscopy with carbon-based nanoparticles for the exploration of the brain extracellular space
      Paviolo, Chiara; Cognet, Laurent
      Neurobiology of Disease (2021), 153 (), 105328CODEN: NUDIEM; ISSN:0969-9961. (Elsevier Inc.)
      A review. Understanding the physiol. and pathol. of the brain requires detailed knowledge of its complex structures as well as dynamic internal processes at very different scales from the macro down to the mol. dimensions. A major yet poorly described brain compartment is the brain extracellular space (ECS). Signalling mols. rapidly diffuse through the brain ECS which is complex and dynamic structure at numerous lengths and time scales. In recent years, characterization of the ECS using nanomaterials has made remarkable progress, including local anal. of nanoscopic dimensions and diffusivity as well as local chem. sensing. In particular, carbon nanomaterials combined with advanced optical technologies, biochem. and biophys. anal., offer novel promises for understanding the ECS morphol. as well as neuron connectivity and neurochem. In this review, we present the state-of-the-art in this quest, which mainly focuses on a type of carbon nanomaterial, single walled carbon nanotubes, as fluorescent nanoprobes to unveil the ECS features in the nanometer domain.
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    10. 10
      Welsher, K.; Liu, Z.; Sherlock, S. P.; Robinson, J. T.; Chen, Z.; Daranciang, D.; Dai, H. A Route to Brightly Fluorescent Carbon Nanotubes for Near-Infrared Imaging in Mice. Nat. Nanotechnol. 2009, 4 (11), 773– 780,  DOI: 10.1038/nnano.2009.294
      10
      A route to brightly fluorescent carbon nanotubes for near-infrared imaging in mice
      Welsher, Kevin; Liu, Zhuang; Sherlock, Sarah P.; Robinson, Joshua Tucker; Chen, Zhuo; Daranciang, Dan; Dai, Hongjie
      Nature Nanotechnology (2009), 4 (11), 773-780CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
      The near-IR photoluminescence intrinsic to semiconducting single-walled carbon nanotubes is ideal for biol. imaging owing to the low autofluorescence and deep tissue penetration in the near-IR region beyond 1 μm. However, biocompatible single-walled carbon nanotubes with high quantum yield have been elusive. Here, we show that sonicating single-walled carbon nanotubes with sodium cholate, followed by surfactant exchange to form phospholipid-polyethylene glycol coated nanotubes, produces in vivo imaging agents that are both bright and biocompatible. The exchange procedure is better than directly sonicating the tubes with the phospholipid-polyethylene glycol, because it results in less damage to the nanotubes and improves the quantum yield. We show whole-animal in vivo imaging using an InGaAs camera in the 1-1.7 μm spectral range by detecting the intrinsic near-IR photoluminescence of the exchange' single-walled carbon nanotubes at a low dose (17 mg l-1 injected dose). The deep tissue penetration and low autofluorescence background allowed high-resoln. intravital microscopy imaging of tumor vessels beneath thick skin.
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    11. 11
      Kruss, S.; Landry, M. P.; Vander Ende, E.; Lima, B. M. A.; Reuel, N. F.; Zhang, J.; Nelson, J.; Mu, B.; Hilmer, A.; Strano, M. Neurotransmitter Detection Using Corona Phase Molecular Recognition on Fluorescent Single-Walled Carbon Nanotube Sensors. J. Am. Chem. Soc. 2014, 136 (2), 713– 724,  DOI: 10.1021/ja410433b
      11
      Neurotransmitter Detection Using Corona Phase Molecular Recognition on Fluorescent Single-Walled Carbon Nanotube Sensors
      Kruss, Sebastian; Landry, Markita P.; Vander Ende, Emma; Lima, Barbara M. A.; Reuel, Nigel F.; Zhang, Jingqing; Nelson, Justin; Mu, Bin; Hilmer, Andrew; Strano, Michael
      Journal of the American Chemical Society (2014), 136 (2), 713-724CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Temporal and spatial changes in neurotransmitter concns. are central to information processing in neural networks. Therefore, biosensors for neurotransmitters are essential tools for neuroscience. The authors applied a new technique, corona phase mol. recognition (CoPhMoRe), to identify adsorbed polymer phases on fluorescent single-walled carbon nanotubes (SWCNTs) that allow for the selective detection of specific neurotransmitters, including dopamine. The authors functionalized and suspended SWCNTs with a library of different polymers (n = 30) contg. phospholipids, nucleic acids, and amphiphilic polymers to study how neurotransmitters modulate the resulting band gap, near-IR (nIR) fluorescence of the SWCNT. The authors identified several corona phases that enable the selective detection of neurotransmitters. Catecholamines such as dopamine increased the fluorescence of specific single-stranded DNA- and RNA-wrapped SWCNTs by 58-80% upon addn. of 100 μM dopamine depending on the SWCNT chirality (n,m). In soln., the limit of detection was 11 nM [Kd = 433 nM for (GT)15 DNA-wrapped SWCNTs]. Mechanistic studies revealed that this turn-on response is due to an increase in fluorescence quantum yield and not covalent modification of the SWCNT or scavenging of reactive oxygen species. When immobilized on a surface, the fluorescence intensity of a single DNA- or RNA-wrapped SWCNT is enhanced by a factor of up to 5.39±1.44, whereby fluorescence signals are reversible. The authors' findings indicate that certain DNA/RNA coronae act as conformational switches on SWCNTs, which reversibly modulate the SWCNT fluorescence. These findings suggest that the authors' polymer-SWCNT constructs can act as fluorescent neurotransmitter sensors in the tissue-compatible nIR optical window, which may find applications in neuroscience.
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    12. 12
      Godin, A. G.; Varela, J. A.; Gao, Z.; Danné, N.; Dupuis, J. P.; Lounis, B.; Groc, L.; Cognet, L. Single-Nanotube Tracking Reveals the Nanoscale Organization of the Extracellular Space in the Live Brain. Nat. Nanotechnol. 2017, 12 (3), 238– 243,  DOI: 10.1038/nnano.2016.248
      12
      Single-nanotube tracking reveals the nanoscale organization of the extracellular space in the live brain
      Godin, Antoine G.; Varela, Juan A.; Gao, Zhenghong; Danne, Noemie; Dupuis, Julien P.; Lounis, Brahim; Groc, Laurent; Cognet, Laurent
      Nature Nanotechnology (2017), 12 (3), 238-243CODEN: NNAABX; ISSN:1748-3387. (Nature Publishing Group)
      The brain is a dynamic structure with the extracellular space (ECS) taking up almost a quarter of its vol. Signaling mols., neurotransmitters and nutrients transit via the ECS, which constitutes a key microenvironment for cellular communication and the clearance of toxic metabolites. The spatial organization of the ECS varies during sleep, development and aging and is probably altered in neuropsychiatric and degenerative diseases, as inferred from electron microscopy and macroscopic biophys. studies. Here the authors show an approach to directly observe the local ECS structures and rheol. in brain tissue using super-resoln. imaging. The authors inject single-walled carbon nanotubes into rat cerebroventricles and follow the near-IR emission of individual nanotubes as they diffuse inside the ECS for tens of minutes in acute slices. Because of the interplay between the nanotube geometry and the ECS local environment, the authors can ext. information about the dimensions and local viscosity of the ECS. A striking diversity of ECS dimensions down to 40 nm and of local viscosity values were found. Moreover, by chem. altering the extracellular matrix of the brains of live animals before nanotube injection, the authors reveal that the rheol. properties of the ECS are affected, but these alterations are local and inhomogeneous at the nanoscale.
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    13. 13
      Paviolo, C.; Soria, F. N.; Ferreira, J. S.; Lee, A.; Groc, L.; Bezard, E.; Cognet, L. Nanoscale Exploration of the Extracellular Space in the Live Brain by Combining Single Carbon Nanotube Tracking and Super-Resolution Imaging Analysis. Methods 2020, 174, 91– 99,  DOI: 10.1016/j.ymeth.2019.03.005
      13
      Nanoscale exploration of the extracellular space in the live brain by combining single carbon nanotube tracking and super-resolution imaging analysis
      Paviolo, Chiara; Soria, Federico N.; Ferreira, Joana S.; Lee, Antony; Groc, Laurent; Bezard, Erwan; Cognet, Laurent
      Methods (Amsterdam, Netherlands) (2020), 174 (), 91-99CODEN: MTHDE9; ISSN:1046-2023. (Elsevier B.V.)
      The brain extracellular space (ECS) is a system of narrow compartments whose intricate nanometric structure has remained elusive until very recently. Understanding such a complex organization represents a technol. challenge that requires a technique able to resolve these nanoscopic spaces and simultaneously characterize their rheol. properties. We recently used single-walled carbon nanotubes (SWCNTs) as near-IR fluorescent probes to map with nanoscale precision the local organization and rheol. of the ECS. Here we expand our method by tracking single nanotubes through super-resoln. imaging in rat organotypic hippocampal slices and acute brain slices from adult mice, pioneering the exploration of the adult brain ECS at the nanoscale. We found a highly heterogeneous ECS, where local rheol. properties can change drastically within few nanometers. Our results suggest differences in local ECS diffusion environments in organotypic slices when compared to adult mouse slices. Data obtained from super-resolved maps of the SWCNT trajectories indicate that ECS widths may vary between brain tissue models, with a looser, less crowded nano-environment in organotypic cultured slices.
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    14. 14
      Tønnesen, J.; Inavalli, V. V. G. K.; Nägerl, U. V. Super-Resolution Imaging of the Extracellular Space in Living Brain Tissue. Cell 2018, 172 (5), 1108– 1121,  DOI: 10.1016/j.cell.2018.02.007
      14
      Super-Resolution Imaging of the Extracellular Space in Living Brain Tissue
      Tonnesen Jan; Inavalli V V G Krishna; Nagerl U Valentin
      Cell (2018), 172 (5), 1108-1121.e15 ISSN:.
      The extracellular space (ECS) of the brain has an extremely complex spatial organization, which has defied conventional light microscopy. Consequently, despite a marked interest in the physiological roles of brain ECS, its structure and dynamics remain largely inaccessible for experimenters. We combined 3D-STED microscopy and fluorescent labeling of the extracellular fluid to develop super-resolution shadow imaging (SUSHI) of brain ECS in living organotypic brain slices. SUSHI enables quantitative analysis of ECS structure and reveals dynamics on multiple scales in response to a variety of physiological stimuli. Because SUSHI produces sharp negative images of all cellular structures, it also enables unbiased imaging of unlabeled brain cells with respect to their anatomical context. Moreover, the extracellular labeling strategy greatly alleviates problems of photobleaching and phototoxicity associated with traditional imaging approaches. As a straightforward variant of STED microscopy, SUSHI provides unprecedented access to the structure and dynamics of live brain ECS and neuropil.
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    15. 15
      Soria, F. N.; Paviolo, C.; Doudnikoff, E.; Arotcarena, M.-L.; Lee, A.; Danné, N.; Mandal, A. K.; Gosset, P.; Dehay, B.; Groc, L.; Cognet, L.; Bezard, E. Synucleinopathy Alters Nanoscale Organization and Diffusion in the Brain Extracellular Space through Hyaluronan Remodeling. Nat. Commun. 2020, 11 (1), 3440,  DOI: 10.1038/s41467-020-17328-9
      15
      Synucleinopathy alters nanoscale organization and diffusion in the brain extracellular space through hyaluronan remodeling
      Soria, Federico N.; Paviolo, Chiara; Doudnikoff, Evelyne; Arotcarena, Marie-Laure; Lee, Antony; Danne, Noemie; Mandal, Amit Kumar; Gosset, Philippe; Dehay, Benjamin; Groc, Laurent; Cognet, Laurent; Bezard, Erwan
      Nature Communications (2020), 11 (1), 3440CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      Abstr.: In recent years, exploration of the brain extracellular space (ECS) has made remarkable progress, including nanoscopic characterizations. However, whether ECS precise conformation is altered during brain pathol. remains unknown. Here we study the nanoscale organization of pathol. ECS in adult mice under degenerative conditions. Using electron microscopy in cryofixed tissue and single nanotube tracking in live brain slices combined with super-resoln. imaging anal., we find enlarged ECS dimensions and increased nanoscale diffusion after α-synuclein-induced neurodegeneration. These animals display a degraded hyaluronan matrix in areas close to reactive microglia. Furthermore, exptl. hyaluronan depletion in vivo reduces dopaminergic cell loss and α-synuclein load, induces microgliosis and increases ECS diffusivity, highlighting hyaluronan as diffusional barrier and local tissue organizer. These findings demonstrate the interplay of ECS, extracellular matrix and glia in pathol., unraveling ECS features relevant for the α-synuclein propagation hypothesis and suggesting matrix manipulation as a disease-modifying strategy.
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    16. 16
      Lim, D.; Ford, T. N.; Chu, K. K.; Mertz, J. Optically Sectioned in Vivo Imaging with Speckle Illumination HiLo Microscopy. J. Biomed. Opt. 2011, 16 (1), 016014– 016014–016018,  DOI: 10.1117/1.3528656
      16
      Optically sectioned in vivo imaging with speckle illumination HiLo microscopy
      Lim Daryl; Ford Tim N; Chu Kengyeh K; Mertz Jerome
      Journal of biomedical optics (2011), 16 (1), 016014 ISSN:.
      We present a simple wide-field imaging technique, called HiLo microscopy, that is capable of producing optically sectioned images in real time, comparable in quality to confocal laser scanning microscopy. The technique is based on the fusion of two raw images, one acquired with speckle illumination and another with standard uniform illumination. The fusion can be numerically adjusted, using a single parameter, to produce optically sectioned images of varying thicknesses with the same raw data. Direct comparison between our HiLo microscope and a commercial confocal laser scanning microscope is made on the basis of sectioning strength and imaging performance. Specifically, we show that HiLo and confocal 3-D imaging of a GFP-labeled mouse brain hippocampus are comparable in quality. Moreover, HiLo microscopy is capable of faster, near video rate imaging over larger fields of view than attainable with standard confocal microscopes. The goal of this paper is to advertise the simplicity, robustness, and versatility of HiLo microscopy, which we highlight with in vivo imaging of common model organisms including planaria, C. elegans, and zebrafish.
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    17. 17
      Lim, D.; Chu, K. K.; Mertz, J. Wide-Field Fluorescence Sectioning with Hybrid Speckle and Uniform-Illumination Microscopy. Opt. Lett. 2008, 33 (16), 1819– 1821,  DOI: 10.1364/OL.33.001819
      17
      Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy
      Lim Daryl; Chu Kengyeh K; Mertz Jerome
      Optics letters (2008), 33 (16), 1819-21 ISSN:0146-9592.
      We describe a method of obtaining optical sectioning with a standard wide-field fluorescence microscope. The method involves acquiring two images, one with nonuniform illumination (in our case, speckle) and another with uniform illumination (in our case, randomized speckle). An evaluation of the local contrast in the speckle-illumination image provides an optically sectioned image with low resolution. This is complemented with high-resolution information obtained from the uniform-illumination image. A fusion of both images leads to a full resolution image that is optically sectioned across all spatial frequencies. This hybrid illumination method is fast, robust, and generalizable to a variety of illumination and imaging configurations.
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    18. 18
      Gao, Z.; Danné, N.; Godin, A. G.; Lounis, B.; Cognet, L. Evaluation of Different Single-Walled Carbon Nanotube Surface Coatings for Single-Particle Tracking Applications in Biological Environments. Nanomaterials 2017, 7 (11), 393,  DOI: 10.3390/nano7110393
      18
      Evaluation of different single-walled carbon nanotube surface coatings for single-particle tracking applications in biological environments
      Gao, Zhenghong; Danne, Noemie; Godin, Antoine Guillaume; Lounis, Brahim; Cognet, Laurent
      Nanomaterials (2017), 7 (11), 393/1-393/12CODEN: NANOKO; ISSN:2079-4991. (MDPI AG)
      Fluorescence imaging of biol. systems down to the single-mol. level has generated many advances in cellular biol. For applications within intact tissue, single-walled carbon nanotubes (SWCNTs) are emerging as distinctive single-mol. nanoprobes, due to their near-IR photoluminescence properties. For this, SWCNT surfaces must be coated using adequate mol. moieties. Yet, the choice of the suspension agent is crit. since it influences both the chem. and emission properties of the SWCNTs within their environment. Here, we compare the most commonly used surface coatings for encapsulating photoluminescent SWCNTs in the context of bio-imaging applications. To be applied as single-mol. nanoprobes, encapsulated nanotubes should display low cytotoxicity, and minimal unspecific interactions with cells while still being highly luminescent so as to be imaged and tracked down to the single nanotube level for long periods of time. We tested the cell proliferation and cellular viability of each surface coating and evaluated the impact of the biocompatible surface coatings on nanotube photoluminescence brightness. Our study establishes that phospholipid-polyethylene glycol-coated carbon nanotube is the best current choice for single nanotube tracking expts. in live biol. samples.
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      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXptlWitQ%253D%253D&md5=f327580a5eba1de807355e70c30c0376
    19. 19
      Danné, N.; Godin, A. G.; Gao, Z.; Varela, J. A.; Groc, L.; Lounis, B.; Cognet, L. Comparative Analysis of Photoluminescence and Upconversion Emission from Individual Carbon Nanotubes for Bioimaging Applications. ACS Photonics 2018, 5 (2), 359– 364,  DOI: 10.1021/acsphotonics.7b01311
      19
      Comparative Analysis of Photoluminescence and Upconversion Emission from Individual Carbon Nanotubes for Bioimaging Applications
      Danne, Noemie; Godin, Antoine G.; Gao, Zhenghong; Varela, Juan A.; Groc, Laurent; Lounis, Brahim; Cognet, Laurent
      ACS Photonics (2018), 5 (2), 359-364CODEN: APCHD5; ISSN:2330-4022. (American Chemical Society)
      Luminescent single-walled carbon nanotubes (SWCNTs) are unique nanoemitters that allow near-IR single-mol. detection within biol. tissues. Interestingly, the recent discovery of upconversion luminescence from (6,5) SWCNTs provides a novel opportunity for deep tissue single SWCNT detection. Yet, the optimal excitation strategy for video-rate imaging of individual SWCNTs within live tissues needs to be detd. taking into account the constraints imposed by the biol. matter. Here, the authors directly compare the luminescence efficiencies of single (6,5) SWCNTs excited by continuous-wave lasers at their second-order excitonic transition, at their K-momentum exciton-phonon sideband, or through upconversion. For these three excitations spanning visible to near-IR wavelengths, the relevance of single SWCNT imaging is considered inside brain tissue. The effects of tissue scattering, absorption, autofluorescence, and temp. increase induced by excitation light are systematically examd.
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    20. 20
      Fakhri, N.; MacKintosh, F. C.; Lounis, B.; Cognet, L.; Pasquali, M. Brownian Motion of Stiff Filaments in a Crowded Environment. Science 2010, 330 (6012), 1804– 1807,  DOI: 10.1126/science.1197321
      20
      Brownian Motion of Stiff Filaments in a Crowded Environment
      Fakhri, Nikta; MacKintosh, Frederick C.; Lounis, Brahim; Cognet, Laurent; Pasquali, Matteo
      Science (Washington, DC, United States) (2010), 330 (6012), 1804-1807CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      The thermal motion of stiff filaments in a crowded environment is highly constrained and anisotropic; it underlies the behavior of such disparate systems as polymer materials, nanocomposites, and the cell cytoskeleton. Despite decades of theor. study, the fundamental dynamics of such systems remains a mystery. Using near-IR video microscopy, we studied the thermal diffusion of individual single-walled carbon nanotubes (SWNTs) confined in porous agarose networks. We found that even a small bending flexibility of SWNTs strongly enhances their motion: The rotational diffusion const. is proportional to the filament-bending compliance and is independent of the network pore size. The interplay between crowding and thermal bending implies that the notion of a filament's stiffness depends on its confinement. Moreover, the mobility of SWNTs and other inclusions can be controlled by tailoring their stiffness.
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    21. 21
      Oudjedi, L.; Parra-Vasquez, A. N. G.; Godin, A. G.; Cognet, L.; Lounis, B. Metrological Investigation of the (6,5) Carbon Nanotube Absorption Cross Section. J. Phys. Chem. Lett. 2013, 4 (9), 1460– 1464,  DOI: 10.1021/jz4003372
      21
      Metrological Investigation of the (6,5) Carbon Nanotube Absorption Cross Section
      Oudjedi, Laura; Parra-Vasquez, A. Nicholas G.; Godin, Antoine G.; Cognet, Laurent; Lounis, Brahim
      Journal of Physical Chemistry Letters (2013), 4 (9), 1460-1464CODEN: JPCLCD; ISSN:1948-7185. (American Chemical Society)
      Using single-nanotube absorption microscopy, the authors measured the absorption cross section of (6,5) C nanotubes at their 2nd-order optical transition. The authors obtained a value of 3.2 × 10-17 cm2/C atom with a precision of 15% and an accuracy <20%. This constitutes the 1st metrol. study of the absorption cross section of chirality-identified nanotubes. Correlative absorption-luminescence microscopies performed on long nanotubes reveal a direct manifestation of exciton diffusion in the nanotube.
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    22. 22
      De Simoni, A.; Griesinger, C. B.; Edwards, F. A. Development of Rat CA1 Neurones in Acute versus Organotypic Slices: Role of Experience in Synaptic Morphology and Activity. J. Physiol. 2003, 550 (1), 135– 147,  DOI: 10.1113/jphysiol.2003.039099
      22
      Development of rat CA1 neurones in acute versus organotypic slices: Role of experience in synaptic morphology and activity
      De Simoni, Anna; Griesinger, Claudius B.; Edwards, Frances A.
      Journal of Physiology (Cambridge, United Kingdom) (2003), 550 (1), 135-147CODEN: JPHYA7; ISSN:0022-3751. (Cambridge University Press)
      Despite their wide use, the physiol. relevance of organotypic slices remains controversial. Such cultures are prepd. at 5 days postnatal. Although some local circuitry remains intact, they develop subsequently in isolation from the animal and hence without plasticity due to experience. Development of synaptic connectivity and morphol. might be expected to proceed differently under these conditions than in a behaving animal. To address these questions, patch-clamp techniques and confocal microscopy were used in the CA1 region of the rat hippocampus to compare acute slices from the third postnatal week with various stages of organotypic slices. Acute slices prepd. at postnatal days (P) 14, 17 and 21 were found to be developmentally equiv. to organotypic slices cultured for 1, 2 and 3 wk, resp., in terms of development of synaptic transmission and dendritic morphol. The frequency of inhibitory and excitatory miniature synaptic currents increased in parallel. Development of dendritic length and primary branching as well as spine d. and proportions of different spine types were also similar in both prepns., at these corresponding stages. The most notable difference between organotypic and acute slices was a four- to five-fold increase in the abs. frequency of glutamatergic (but not GABAergic) miniature postsynaptic currents in organotypic slices. This was probably related to an increase in complexity of higher order dendritic branching in organotypic slices, as measured by fractal anal., resulting in an increased total synapse no. Both increased excitatory miniature synaptic current frequency and dendritic complexity were already established during the first week in culture. The level of complexity then stayed const. in both prepns. over subsequent stages, with synaptic frequency increasing in parallel. Thus, although connectivity was greater in organotypic slices, once this was established, development continued in both prepns. at a remarkably similar rate. We conclude that, for the parameters studied, changes seem to be preprogrammed by 5 days and their subsequent development is largely independent of environment.
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    23. 23
      Vargová, L.; Syková, E. Astrocytes and Extracellular Matrix in Extrasynaptic Volume Transmission. Philos. Trans. R. Soc. London, B, Biol. Sci. 2014, 369 (1654), 20130608,  DOI: 10.1098/rstb.2013.0608
      23
      Astrocytes and extracellular matrix in extrasynaptic volume transmission
      Vargova Lydia; Sykova Eva
      Philosophical transactions of the Royal Society of London. Series B, Biological sciences (2014), 369 (1654), 20130608 ISSN:.
      Volume transmission is a form of intercellular communication that does not require synapses; it is based on the diffusion of neuroactive substances across the brain extracellular space (ECS) and their binding to extrasynaptic high-affinity receptors on neurons or glia. Extracellular diffusion is restricted by the limited volume of the ECS, which is described by the ECS volume fraction α, and the presence of diffusion barriers, reflected by tortuosity λ, that are created, for example, by fine astrocytic processes or extracellular matrix (ECM) molecules. Organized astrocytic processes, ECM scaffolds or myelin sheets channel the extracellular diffusion so that it is facilitated in a certain direction, i.e. anisotropic. The diffusion properties of the ECS are profoundly influenced by various processes such as the swelling and morphological rebuilding of astrocytes during either transient or persisting physiological or pathological states, or the remodelling of the ECM in tumorous or epileptogenic tissue, during Alzheimer's disease, after enzymatic treatment or in transgenic animals. The changing diffusion properties of the ECM influence neuron-glia interaction, learning abilities, the extent of neuronal damage and even cell migration. From a clinical point of view, diffusion parameter changes occurring during pathological states could be important for diagnosis, drug delivery and treatment.
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    24. 24
      Piet, R.; Vargová, L.; Syková, E.; Poulain, D. A.; Oliet, S. H. R. Physiological Contribution of the Astrocytic Environment of Neurons to Intersynaptic Crosstalk. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (7), 2151– 2155,  DOI: 10.1073/pnas.0308408100
      24
      Physiological contribution of the astrocytic environment of neurons to intersynaptic crosstalk
      Piet, Richard; Vargova, Lydia; Sykova, Eva; Poulain, Dominique A.; Oliet, Stephane H. R.
      Proceedings of the National Academy of Sciences of the United States of America (2004), 101 (7), 2151-2155CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Interactions between sep. synaptic inputs converging on the same target appear to contribute to the fine-tuning of information processing in the central nervous system. Intersynaptic crosstalk is made possible by transmitter spillover from the synaptic cleft and its diffusion over a distance to neighboring synapses. This is the case for glutamate, which inhibits γ-aminobutyric acid (GABA)ergic transmission in several brain regions through the activation of presynaptic receptors. Such heterosynaptic modulation depends on factors that influence diffusion in the extracellular space (ECS). Because glial cells represent a phys. barrier to diffusion and, in addn., are essential for glutamate uptake, the authors investigated the physiol. contribution of the astrocytic environment of neurons to glutamate-mediated intersynaptic communication in the brain. The reduced astrocytic coverage of magnocellular neurons occurring in the supraoptic nucleus of lactating rats facilitates diffusion in the ECS, as revealed by tortuosity and vol. fraction measurements. Under these conditions, glutamate spillover, monitored through metabotropic glutamate receptor-mediated depression of GABAergic transmission, is greatly enhanced. Conversely, impeding diffusion with dextran largely prevents crosstalk between glutamatergic and GABAergic afferent inputs. Astrocytes, therefore, by hindering diffusion in the ECS, regulate intersynaptic communication between neighboring synapses and, probably, overall vol. transmission in the brain.
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.nanolett.1c04259.

    • Movie of raw acquisition of CNTs (AVI)

    • Materials and Methods, including figures (PDF)


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