a-dual-color-fiber-photometry-method-for-recording-astrocyte-neuron

A Dual-Color Fiber Photometry Method for Recording Astrocyte-Neuron Activity Across Multiple Brain Regions During Learning and Memory Behaviors

Learning and memory depend critically on the coordination between neurons and astrocytes across distributed brain regions. Astrocytes modulate synaptic ...

Third Military Medical University

Two-photon Ca imaging has become a popular approach for monitoring neuronal population activity with cellular or subcellular resolution in vivo. This approach allows for the recording of hundreds to thousands of neurons per animal and thus leads to a large amount of data to be processed. In particular, manually drawing regions of interest is the most time-consuming aspect of data analysis. However, the development of automated image analysis pipelines, which will be essential for dealing with the likely future deluge of imaging data, remains a major challenge. To address this issue, we developed NeuroSeg, an open-source MATLAB program that can facilitate the accurate and efficient segmentation of neurons in two-photon Ca imaging data. We proposed an approach using a generalized Laplacian of Gaussian filter to detect cells and weighting-based segmentation to separate individual cells from the background. We tested this approach on an in vivo two-photon Ca imaging dataset obtained from mouse cortical neurons with differently sized view fields. We show that this approach exhibits superior performance for cell detection and segmentation compared with the existing published tools. In addition, we integrated the previously reported, activity-based segmentation into our approach and found that this combined method was even more promising. The NeuroSeg software, including source code and graphical user interface, is freely available and will be a useful tool for in vivo brain activity mapping.

NeuroSeg: automated cell detection and segmentation for in vivo two-photon Ca imaging data.

In the primary auditory cortex (A1), neuronal ensembles are activated relative to anticipated sound events following rhythmic stimulation, but whether the echo responses of the neurons are related to their frequency selectivity remains unknown. Therefore, we used in vivo two-photon Ca imaging to record the neuronal activities in the mouse A1 to elucidate the relationship between their echo responses and frequency selectivity. We confirmed the presence of echo responses in a subgroup of mouse Layer 2/3 A1 neurons following a train of rhythmic pure tone stimulation. After testing with a range of frequencies, we found that these echo responses occurred preferentially close to the best frequencies of the neurons. The local organization of the echo responses of the neurons was heterogeneous in the A1. Therefore, these results indicate that the observed echo responses of neurons within A1 are highly related to their frequency selectivity.

Frequency selectivity of echo responses in the mouse primary auditory cortex.

 two-photon Ca imaging is a powerful tool for recording neuronal activities during perceptual tasks and has been increasingly applied to behaving animals for acute or chronic experiments. However, the auditory cortex is not easily accessible to imaging because of the abundant temporal muscles, arteries around the ears and their lateral locations. Here, we report a protocol for two-photon Ca imaging in the auditory cortex of head-fixed behaving mice. By using a custom-made head fixation apparatus and a head-rotated fixation procedure, we achieved two-photon imaging and in combination with targeted cell-attached recordings of auditory cortical neurons in behaving mice. Using synthetic Ca indicators, we recorded the Ca transients at multiple scales, including neuronal populations, single neurons, dendrites and single spines, in auditory cortex during behavior. Furthermore, using genetically encoded Ca indicators (GECIs), we monitored the neuronal dynamics over days throughout the process of associative learning. Therefore, we achieved two-photon functional imaging at multiple scales in auditory cortex of behaving mice, which extends the tool box for investigating the neural basis of audition-related behaviors.

Two-Photon Functional Imaging of the Auditory Cortex in Behaving Mice: From Neural Networks to Single Spines.

The ability to remember and to navigate to safe places is necessary for survival. Place navigation is known to involve medial entorhinal cortex (MEC)-hippocampal connections. However, learning-dependent changes in neuronal activity in the distinct circuits remain unknown. Here, by using optic fiber photometry in freely behaving mice, we discovered the experience-dependent induction of a persistent-task-associated (PTA) activity. This PTA activity critically depends on learned visual cues and builds up selectively in the MEC layer II-dentate gyrus, but not in the MEC layer III-CA1 pathway, and its optogenetic suppression disrupts navigation to the target location. The findings suggest that the visual system, the MEC layer II, and the dentate gyrus are essential hubs of a memory circuit for visually guided navigation.

A Visual-Cue-Dependent Memory Circuit for Place Navigation.

The sensory responses of cortical neuronal populations following training have been extensively studied. However, the spike firing properties of individual cortical neurons following training remain unknown. Here, we have combined two-photon Ca imaging and single-cell electrophysiology in awake behaving mice following auditory associative training. We find a sparse set (~5%) of layer 2/3 neurons in the primary auditory cortex, each of which reliably exhibits high-rate prolonged burst firing responses to the trained sound. Such bursts are largely absent in the auditory cortex of untrained mice. Strikingly, in mice trained with different multitone chords, we discover distinct subsets of neurons that exhibit bursting responses specifically to a chord but neither to any constituent tone nor to the other chord. Thus, our results demonstrate an integrated representation of learned complex sounds in a small subset of cortical neurons.

Single-neuron representation of learned complex sounds in the auditory cortex.

Memory persistence is a fundamental cognitive process for guiding behaviors and is considered to rely mostly on neuronal and synaptic plasticity. Whether and how astrocytes contribute to memory persistence is largely unknown. Here, by using two-photon Ca imaging in head-fixed mice and fiber photometry in freely moving mice, we show that aversive sensory stimulation activates α7-nicotinic acetylcholine receptors (nAChRs) in a subpopulation of astrocytes in the auditory cortex. We demonstrate that fear learning causes the de novo induction of sound-evoked Ca transients in these astrocytes. The astrocytic responsiveness persisted over days along with fear memory and disappeared in animals that underwent extinction of learned freezing behavior. Conditional genetic deletion of α7-nAChRs in astrocytes significantly impaired fear memory persistence. We conclude that learning-acquired, α7-nAChR-dependent astrocytic responsiveness is an integral part of the cellular substrate underlying memory persistence.

Fear learning induces α7-nicotinic acetylcholine receptor-mediated astrocytic responsiveness that is required for memory persistence.

Astrocytic Ca transients are essential for astrocyte integration into neural circuits. These Ca transients are primarily sequestered in subcellular domains, including primary branches, branchlets and leaflets, and endfeet. In previous studies, it suggests that aging causes functional defects in astrocytes. Until now, it was unclear whether and how aging affects astrocytic Ca transients at subcellular domains. In this study, we combined a genetically encoded Ca sensor (GCaMP6f) and  two-photon Ca imaging to determine changes in Ca transients within astrocytic subcellular domains during brain aging. We showed that aging increased Ca transients in astrocytic primary branches, higher-order branchlets, and terminal leaflets. However, Ca transients decreased within astrocytic endfeet during brain aging, which could be caused by the decreased expressions of Aquaporin-4 (AQP4). In addition, aging-induced changes of Ca transient types were heterogeneous within astrocytic subcellular domains. These results demonstrate that the astrocytic Ca transients within subcellular domains are affected by aging differently. This finding contributes to a better understanding of the physiological role of astrocytes in aging-induced neural circuit degeneration.

Astrocytes exhibit diverse Ca changes at subcellular domains during brain aging.

Quantitative and mechanistic understanding of learning and long-term memory at the level of single neurons in living brains require highly demanding techniques. A specific need is to precisely label one cell whose firing output property is pinpointed amidst a functionally characterized large population of neurons through the learning process and then investigate the distribution and properties of dendritic inputs. Here, we disseminate an integrated method of daily two-photon neuronal population Ca imaging through an auditory associative learning course, followed by targeted single-cell loose-patch recording and electroporation of plasmid for enhanced chronic Ca imaging of dendritic spines in the targeted cell. Our method provides a unique solution to the demand, opening a solid path toward the hard-cores of how learning and long-term memory are physiologically carried out at the level of single neurons and synapses.

Daily two-photon neuronal population imaging with targeted single-cell electrophysiology and subcellular imaging in auditory cortex of behaving mice.

The studying of rapid neuronal signaling across large spatial scales in intact, living brains requires both high temporal resolution and versatility of the measurement device.

Ten-kilohertz two-photon microscopy imaging of single-cell dendritic activity and hemodynamics .

Neocortical layer 6 (L6) is less understood than other more superficial layers, largely owing to limitations of performing high-resolution investigations . Here, we show that labeling with the Challenge Virus Standard (CVS) rabies virus strain enables high-quality imaging of L6 neurons by conventional two-photon microscopes. CVS virus injection into the medial geniculate body can selectively label L6 neurons in the auditory cortex. Only three days after injection, dendrites and cell bodies of L6 neurons could be imaged across all cortical layers. Ca imaging in awake mice showed that sound stimulation evokes neuronal responses from cell bodies with minimal contamination from neuropil signals. In addition, dendritic Ca imaging revealed significant responses from spines and trunks across all layers. These results demonstrate a reliable method capable of rapid, high-quality labeling of L6 neurons that can be readily extended to other brain regions.

Rabies virus-based labeling of layer 6 corticothalamic neurons for two-photon imaging .

The sensory neocortex has been suggested to be a substrate for long-term memory storage, yet which exact single cells could be specific candidates underlying such long-term memory storage remained neither known nor visible for over a century. Here, using a combination of day-by-day two-photon Ca imaging and targeted single-cell loose-patch recording in an auditory associative learning paradigm with composite sounds in male mice, we reveal sparsely distributed neurons in layer 2/3 of auditory cortex emerged step-wise from quiescence into bursting mode, which then invariably expressed holistic information of the learned composite sounds, referred to as holistic bursting (HB) cells. Notably, it was not shuffled populations but the same sparse HB cells that embodied the behavioral relevance of the learned composite sounds, pinpointing HB cells as physiologically-defined single-cell candidates of an engram underlying long-term memory storage in auditory cortex.

Holistic bursting cells store long-term memory in auditory cortex.

Postsynaptic currents of chemical synapse are modulated by multitudinous neurotransmitters, such as acetylcholine, dopamine, glutamate, and γ-aminobutyric acid, many of which have been used in the treatment of neurological diseases. Here, based on molecular dynamics simulations and quantum chemical calculation, we propose that 30- to 45-THz photons can resonate with a variety of typical neurotransmitter molecules and make them absorb photon energy to activate the transition to high energy state, which is expected to be a new method of neural regulation. Furthermore, we verified the calculated results through experiments that THz irradiation could substantively change neuronal signal emission and enhance the frequency, amplitude, and dynamic properties of excitatory postsynaptic current and inhibitory postsynaptic current. In addition, we demonstrated the potential of neural information regulation by THz photons through 2-photon imaging in vivo. These findings are expected to improve the understanding of the physical mechanism of biological phenomena and facilitate the application of terahertz technology in neural regulation and the development of new functional materials.

Neuromodulation of Chemical Synaptic Transmission Driven by THz Photons.

Resilience measures generally are not health specific, nor do they account for the multiple dimensions required for individuals to overcome health challenges. To bridge this gap, we developed and validated a multi-dimensional Health Resilience Scale (HRS) for community-dwelling adults in Singapore.

Development and validation of the multi-dimensional health resilience scale for community-dwelling adults.

Previous studies based on layer specificity suggest that ascending signals from the thalamus to the sensory neocortex preserve spatially organized information, but it remains unknown whether sensory information descending from sensory neocortex to the thalamus also maintains such spatial organization pattern. By focusing on projection specificity, we mapped the tone response properties of two groups of cortical neurons in the primary auditory cortex (A1), based on the relationship between their specific connections to other regions and their function in ascending (thalamocortical recipient [TR] neurons) or descending (corticothalamic [CT] neurons) auditory information. A clear tonotopic gradient was observed among TR neurons, but not CT neurons. Additionally, CT neurons exhibited markedly higher heterogeneity in their frequency tuning and had broader bandwidth than TR neurons. These results reveal that the information flow descending from A1 to the thalamus via CT neurons is not arranged tonotopically, suggesting that the descending information flow possibly contributes to higher-order feedback processing of diverse auditory inputs.

Article	Year
NeuroSeg: automated cell detection and segmentation for in vivo two-photon Ca imaging data. Brain structure & function\| PubMed ID: 29124351	2018
Frequency selectivity of echo responses in the mouse primary auditory cortex. Scientific reports\| PubMed ID: 29311673	2018
Two-Photon Functional Imaging of the Auditory Cortex in Behaving Mice: From Neural Networks to Single Spines. Frontiers in neural circuits\| PubMed ID: 29740289	2018
A Visual-Cue-Dependent Memory Circuit for Place Navigation. Neuron\| PubMed ID: 29909996	2018
Single-neuron representation of learned complex sounds in the auditory cortex. Nature communications\| PubMed ID: 32868773	2020
Fear learning induces α7-nicotinic acetylcholine receptor-mediated astrocytic responsiveness that is required for memory persistence. Nature neuroscience\| PubMed ID: 34782794	2021
Astrocytes exhibit diverse Ca changes at subcellular domains during brain aging. Frontiers in aging neuroscience\| PubMed ID: 36389078	2022
Daily two-photon neuronal population imaging with targeted single-cell electrophysiology and subcellular imaging in auditory cortex of behaving mice. Frontiers in cellular neuroscience\| PubMed ID: 36937184	2023
Ten-kilohertz two-photon microscopy imaging of single-cell dendritic activity and hemodynamics . Neurophotonics\| PubMed ID: 37152357	2023
Rabies virus-based labeling of layer 6 corticothalamic neurons for two-photon imaging . iScience\| PubMed ID: 37250327	2023
Holistic bursting cells store long-term memory in auditory cortex. Nature communications\| PubMed ID: 38062015	2023
Neuromodulation of Chemical Synaptic Transmission Driven by THz Photons. Research (Washington, D.C.)\| PubMed ID: 39285946	2022
Development and validation of the multi-dimensional health resilience scale for community-dwelling adults. Frontiers in public health\| PubMed ID: 40013045	2025
Tonotopy is not preserved in a descending stage of auditory cortex. eLife\| PubMed ID: 41143504	2025

Ruijie Li

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