Synaptic responses adapt on millisecond-to-second timescales through short-term plasticity (STP), a key process that filters and transforms neuronal information. While STP is classically ascribed to presynaptic release mechanisms, postsynaptic receptor properties-particularly desensitization and surface diffusion-also shape synaptic responses. Here, we dissect pre- and postsynaptic contributions to synaptic adaptation using molecular tools to visualize glutamate release and manipulate AMPA receptor (AMPAR) diffusion in intact circuits. We find that synaptic gain during STP is tuned by synapse-specific regulation of AMPAR biophysics and diffusion-trapping. These features are determined constitutively by auxiliary subunit profiles and dynamically by activity-dependent signaling engaged during long-term plasticity. With modeling, we quantified how short-term synaptic dynamics are impacted by postsynaptic regulation of filtering properties, which broadened heterogeneity of filtering timescales to refine temporal selectivity in synaptic networks. By augmenting desensitization-mediated synaptic depression, AMPAR diffusion-trapping emerges as a fundamental regulatory mechanism of postsynaptic integration and circuit-level information processing.

Synaptic responses adapt to fast repetitive inputs during bursts of neuronal network activity over timescales of milliseconds to seconds, either transiently facilitating or depressing. This high-frequency stimulus-dependent short-term synaptic plasticity (HF-STP) relies on a number of molecular processes that collectively endow synapses with filtering properties for information processing, optimized for the transmission of certain input frequencies and patterns in distinct circuits [1][1]–[3][2]. Changes in HF-STP are traditionally thought to stem from changes in pre-synaptic transmitter release [1][1],[2][3], but post-synaptic modifications in receptor biophysical properties or surface diffusion also regulate HF-STP [4][4]–[11][5]. A major challenge in understanding synapse function is to decipher how pre- and post-synaptic mechanisms synergistically tune synaptic transmission efficacy during HF-STP, and to determine how neuronal activity modifies post-synaptic signal computation and integration to diversify neuronal circuit function. Here, taking advantage of new molecular tools to directly visualize glutamate release [12][6] and specifically manipulate the surface diffusion of endogenous AMPAR in intact circuits [13][7], we define the respective contributions of pre-synaptic glutamate release, AMPAR desensitization and surface mobility to frequency-dependent synaptic adaptation. We demonstrate that post-synaptic gain control and signal integration capacity in synaptic networks is influenced by synapse-specific differences in AMPAR desensitization and diffusion-trapping characteristics that are shaped by molecular signaling events recruited during LTP. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1 [2]: #ref-3 [3]: #ref-2 [4]: #ref-4 [5]: #ref-11 [6]: #ref-12 [7]: #ref-13

The Frontiers in Neurophotonics Symposium is a biennial event that brings together neurobiologists and physicists/engineers who share interest in the development of leading-edge photonics-based approaches to understand and manipulate the nervous system, from its individual molecular components to complex networks in the intact brain. In this Community paper, we highlight several topics that have been featured at the symposium that took place in October 2022 in Québec City, Canada.

Progress in neuroscience research hinges on technical advances in visualizing living brain tissue with high fidelity and facility. Current neuroanatomical imaging approaches either require tissue fixation (electron microscopy), do not have cellular resolution (magnetic resonance imaging) or only give a fragmented view (fluorescence microscopy). Here, we show how regular light microscopy together with fluorescence labeling of the interstitial fluid in the extracellular space provide comprehensive optical access in real-time to the anatomical complexity and dynamics of living brain tissue at submicron scale. Using several common fluorescence microscopy modalities (confocal, light-sheet and 2-photon microscopy) in mouse organotypic and acute brain slices and the intact mouse brain in vivo, we demonstrate the value of this straightforward ‘shadow imaging’ approach by revealing neurons, microglia, tumor cells and blood capillaries together with their complete anatomical tissue contexts. In addition, we provide quantifications of perivascular spaces and the volume fraction of the extracellular space of brain tissue in vivo.

Lattice light-sheet microscopy (LLSM) is a very efficient technique for high resolution 3D imaging of dynamic phenomena in living biological samples. However, LLSM imaging remains limited in depth due to optical aberrations caused by sample-based refractive index mismatch. Here, we propose a simple and low-cost active image optimization (AIO) method to recover high resolution imaging inside thick biological samples. AIO is based on (1) a light-sheet autofocus step (AF) followed by (2) an adaptive optics image-based optimization. We determine the optimum AIO parameters to provide a fast, precise and robust aberration correction on biological samples. Finally, we demonstrate the performances of our approach on sub-micrometric structures in brain slices and plant roots.

Regulation of synaptic neurotransmitter receptor content is a fundamental mechanism for tuning synaptic efficacy during experience-dependent plasticity and behavioral adaptation. However, experimental approaches to track and modify receptor movements in integrated experimental systems are limited. Exploiting AMPA-type glutamate receptors (AMPARs) as a model, we generated a knock-in mouse expressing the biotin acceptor peptide (AP) tag on the GluA2 extracellular N-terminal. Cell-specific introduction of biotin ligase allows the use of monovalent or tetravalent avidin variants to respectively monitor or manipulate the surface mobility of endogenous AMPAR containing biotinylated AP–GluA2 in neuronal subsets. AMPAR immobilization precluded the expression of long-term potentiation and formation of contextual fear memory, allowing target-specific control of the expression of synaptic plasticity and animal behavior. The AP tag knock-in model offers unprecedented access to resolve and control the spatiotemporal dynamics of endogenous receptors, and opens new avenues to study the molecular mechanisms of synaptic plasticity and learning.

Astrocytes regulate neuronal information processing through a variety of spatio-temporal calcium signals. Recent advances in calcium imaging have started to shine light on astrocytic activity, but the complexity and size of the recorded data strongly call for more advanced computational analysis tools. Their development is currently hindered by the lack of reliable, labeled annotations that are essential for the evaluation of algorithms and the training of learning-based methods. To solve this labeling problem, we have designed a generator of 2D/3D lattice light sheet microscopy (LLSM) sequences which realistically depict the calcium dynamics of astrocytes. By closely modeling calcium kinetics in real astrocytic ramifications, the generated datasets open the door for the deployment of convolutional neural networks in LLSM.

Lattice light sheet (LLS) fluorescence microscopy is a powerful recent technique for in vivo imaging of single and multicellular samples at very high spatio-temporal resolutions. We built a LLS microscope in which we added a photostimulation path to perform all-optical neurophysiological studies in rodent hippocampal brain slices. Thanks to the photo-stimulation path we could achieve fluorescence recovery after photobleaching (FRAP) or glutamate uncaging at spatially and temporally controlled regions of interest. Several fluorescence labelling protocols were employed depending on the imaged structure. Sub-micrometric neuronal elements such as spines or dendritic vesicles could be imaged down to ~20 μm below the surface. We demonstrate the performances of LLS in several ongoing studies: measurement of AMPA receptor surface diffusion at single spines, vesicular transport in dendrites, spontaneous and stimulated local calcium activity in neurons and astrocytes.

The advent of scanning two-photon microscopy (2PM) has created a fertile new avenue for noninvasive investigation of brain activity in depth. One principal weakness of this method, however, lies with the limit of scanning speed, which makes optical interrogation of action potential-like activity in a neuronal network problematic. Encoded multisite two-photon microscopy (eMS2PM), a scanless method that allows simultaneous imaging of multiple targets in depth with high temporal resolution, addresses this drawback. eMS2PM uses a liquid crystal spatial light modulator to split a high-power femto-laser beam into multiple subbeams. To distinguish them, a digital micromirror device encodes each subbeam with a specific binary amplitude modulation sequence. Fluorescence signals from all independently targeted sites are then collected simultaneously onto a single photodetector and site-specifically decoded. We demonstrate that eMS2PM can be used to image spike-like voltage transients in cultured cells and fluorescence transients (calcium signals in neurons and red blood cells in capillaries from the cortex) in depth in vivo. These results establish eMS2PM as a unique method for simultaneous acquisition of neuronal network activity.

Uncovering principles that regulate energy metabolism in the brain requires mapping of partial pressure of oxygen (PO(2)) and blood flow with high spatial and temporal resolution. Using two-photon phosphorescence lifetime microscopy (2PLM) and the oxygen probe PtP-C343, we show that PO(2) can be accurately measured in the brain at depths up to 300 μm with micron-scale resolution. In addition, 2PLM allowed simultaneous measurements of blood flow and of PO(2) in capillaries with less than one-second temporal resolution. Using this approach, we detected erythrocyte-associated transients (EATs) in oxygen in the rat olfactory bulb and showed the existence of diffusion-based arterio-venous shunts. Sensory stimulation evoked functional hyperemia, accompanied by an increase in PO(2) in capillaries and by a biphasic PO(2) response in the neuropil, consisting of an 'initial dip' and a rebound. 2PLM of PO(2) opens new avenues for studies of brain metabolism and blood flow regulation.

Low-magnification high-numerical aperture objectives maximize the collection efficiency for scattered two-photon excited fluorescence (2PEF), but non-descanned detection schemes for such objectives demand optical components much bigger than standard microscope optics. Fiber coupling offers the possibility of removing bulky multi-channel detectors from the collection site, but coupling and transmission losses are generally believed to outweigh the benefits of optical fibers. We present here two new developments based on large-core fiber-optic fluorescence detection that illustrate clear advantages over conventional air-coupled 2PEF detection schemes. First, with minimal modifications of a commercial microscope, we efficiently couple the output of a 20×/NA0.95 objective to a large-core liquid light guide and we obtain a 7-fold collection gain when imaging astrocytes at 100 μm depth in acute brain slices of adult ALDH1L1-GFP mice. Second, combining 2PEF microscopy and 4-color detection on a custom microscope, mode scrambling inside a 2-mm plastic optical fiber is shown to cancel out the spatially non-uniform spectral sensitivity observed with air-coupled detectors. Spectral unmixing of images of brainbow mice taken with a fiber-coupled detector revealed a uniform color distribution of hippocampal neurons across a large field of view. Thus, fiber coupling improves both the efficiency and the homogeneity of 2PEF collection.

DOI of original article:10.1016/j.jneumeth.2011.03.015. ∗ Corresponding author at: Laboratorie de Neurophysiologie et Nouvelles Microscopies Universite Paris Descartes, Paris F-75006, France. Tel.: +33 1 42 86 42 21; fax: +33 42 86 42 41 51. E-mail address: martin.oheim@parisdescartes.fr (M. Oheim). 1 Present address: Dept. of Neurophysiology, Brain Research Institute, University of Zurich, Winterhurestrasse 190, CH-8057 Zurich, Switzerland.

L'invention porte sur un procede d'imagerie d'un echantillon cellulaire par une imagerie a multiples photons, le procede comprenant : la disposition d'un faisceau d'excitation multiphotonique ; la separation du faisceau d'excitation multiphotonique en une pluralite de N sous-faisceaux ; la modulation de l'amplitude de la pluralite de N sous-faisceaux pour creer N motifs de modulation temporelle respectifs ; la focalisation de la pluralite de N sous-faisceaux modules en N points focaux separes spatialement respectifs P 1 ... P N a l'interieur de l'echantillon cellulaire ; la detection simultanee de signaux fluorescents emis a partir des N points focaux P 1 ... P N ; et la demodulation des signaux fluorescents detectes de sorte que les donnees de fluorescence relatives aux points focaux respectifs P 1 ... P N peuvent etre obtenues. L'invention porte egalement sur un appareil d'imagerie d'un echantillon cellulaire.

Object Diffusion tensor (DT) imaging tractography is increasingly used to map fiber tracts in patients with surgical brain lesions to reduce the risk of postoperative functional deficit. There are few validation studies of DT imaging tractography in these patients. The aim of this study was to compare DT imaging tractography of language fiber tracts by using intraoperative subcortical electrical stimulations. Methods The authors included 10 patients with low-grade gliomas or dysplasia located in language areas. The MR imaging examination included 3D T1-weighted images for anatomical coregistration, FLAIR, and DT images. Diffusion tensors and fiber tracts were calculated using in-house software. Four tracts were reconstructed in each patient including the arcuate fasciculus, the inferior occipitofrontal fasciculus, and 2 premotor fasciculi (the subcallosal medialis fiber tract and cortical fibers originating from the medial and lateral premotor areas). The authors compared fiber tracts reconstructed using DT imaging with those evidenced using intraoperative subcortical language mapping. Results Seventeen (81%) of 21 positive stimulations were concordant with DT imaging fiber bundles (located within 6 mm of a fiber tract). Four positive stimulations were not located in the vicinity of a DT imaging fiber tract. Stimulations of the arcuate fasciculus mostly induced articulatory and phonemic/syntactic disorders and less frequently semantic paraphasias. Stimulations of the inferior occipitofrontal fasciculus induced semantic paraphasias. Stimulations of the premotor-related fasciculi induced dysarthria and articulatory planning deficit. Conclusions There was a good correspondence between positive stimulation sites and fiber tracts, suggesting that DT imaging fiber tracking is a reliable technique but not yet optimal to map language tracts in patients with brain lesions. Negative tractography does not rule out the persistence of a fiber tract, especially when invaded by the tumor. Stimulations of the different tracts induced variable language disorders that were specific to each fiber tract.

BACKGROUND:The generation of transgenic mice expressing combinations of fluorescent proteins has greatly aided the reporting of activity and identification of specific neuronal populations. Methods capable of separating multiple overlapping fluorescence emission spectra, deep in the living brain, with high sensitivity and temporal resolution are therefore required. Here, we investigate to what extent spectral unmixing addresses these issues.METHODOLOGY/PRINCIPAL FINDINGS:Using fluorescence resonance energy transfer (FRET)-based reporters, and two-photon laser scanning microscopy with synchronous multichannel detection, we report that spectral unmixing consistently improved FRET signal amplitude, both in vitro and in vivo. Our approach allows us to detect odor-evoked FRET transients 180-250 microm deep in the brain, the first demonstration of in vivo spectral imaging and unmixing of FRET signals at depths greater than a few tens of micrometer. Furthermore, we determine the reporter efficiency threshold for which FRET detection is improved by spectral unmixing.CONCLUSIONS/SIGNIFICANCE:Our method allows the detection of small spectral variations in depth in the living brain, which is essential for imaging efficiently transgenic animals expressing combination of multiple fluorescent proteins.

Language processing involves multiple neuronal structures in the human auditory cortex. Although a variety of neuroimaging and mapping techniques have been implemented to better understand language processing at the level of the auditory cortex, much is unknown regarding how and by what pathways these structures interact during essential tasks such as sentence comprehension. In this study, the effective and structural connectivity at the level of the auditory cortex were investigated. First, blood oxygenation level-dependent (BOLD) responses were measured with time-resolved functional magnetic resonance imaging (fMRI) during audition of short sentences. Once BOLD activation maps were obtained, the effective connectivity between primary auditory cortex and the surrounding auditory regions on the supratemporal plane and superior temporal gyrus (STG) were investigated using Granger causality mapping (GCM). Effective connectivity was observed between the primary auditory cortex and (1) the lateral planum polare and anterior STG, and (2) the lateral planum temporale and posterior STG. By using diffusion tensor probabilistic mapping (DTPM), rostral and caudal fiber pathways were detected between regions depicting effective connectivity. The effective and structural connectivity results of the present study provide further insight as to how auditory stimuli (i.e., human language) is processed at the level of the auditory cortex. Furthermore, combining BOLD fMRI-based GCM and DTPM analysis could provide a novel means to study effective and structural connectivity not only in the auditory cortex, but also in other cortical regions.