Monitoring astrocytic Ca²⁺ activity is essential to understand the physiological and pathological roles of astrocytes in the brain. However, previous commonly used methods for studying astrocytic Ca²⁺ activities can be applied in only anesthetized or head-fixed animals, which significantly affects in vivo astrocytic Ca²⁺ dynamics. In the current study, we combined optic fiber recordings with genetically encoded Ca²⁺ indicators (GECIs) to monitor astrocytic activity in freely behaving mice. This approach enabled selective and reliable measurement of astrocytic Ca²⁺ activity, which was verified by the astrocyte-specific labeling of GECIs and few movement artifacts. Additionally, astrocytic Ca²⁺ activities induced by locomotion or footshock were stably recorded in the cortices and hippocampi of freely behaving mice. Furthermore, this method allowed for the longitudinal study of astrocytic activities over several weeks. This work provides a powerful approach to record astrocytic activity selectively, stably, and chronically in freely behaving mice.

Aging causes astrocyte morphological degeneration and functional deficiency, which impairs neuronal functions. Until now, whether age-induced neuronal deficiency could be alleviated by engraftment of glial progenitor cell (GPC) derived astrocytes remained unknown. In the current study, GPCs were generated from embryonic cortical neural stem cells in vitro and transplanted into the brains of aged mice. Their integration and intervention effects in the aged brain were examined 12 months after transplantation. Results indicated that these in-vitro-generated GPC-derived astrocytes possessed normal functional properties. After transplantation they could migrate, differentiate, achieve long-term integration, and maintain much younger morphology in the aged brain. Additionally, these GPC-derived astrocytes established endfeet expressing aquaporin-4 (AQP4) and ameliorate AQP4 polarization in the aged neocortex. More importantly, age-dependent sensory response degeneration was reversed by GPC transplantation. This work demonstrates that rejuvenation of the astrocyte niche is a promising treatment to prevent age-induced degradation of neuronal and behavioral functions.

Astrocytic Ca²⁺transients are essential for astrocyte integration into neural circuits. These Ca²⁺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 Ca²⁺transients at subcellular domains. In this study, we combined a genetically encoded Ca²⁺sensor (GCaMP6f) andin vivotwo-photon Ca²⁺imaging to determine changes in Ca²⁺transients within astrocytic subcellular domains during brain aging. We showed that aging increased Ca²⁺transients in astrocytic primary branches, higher-order branchlets, and terminal leaflets. However, Ca²⁺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 Ca²⁺transient types were heterogeneous within astrocytic subcellular domains. These results demonstrate that the astrocytic Ca²⁺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.

The astrocyte-neuron network in the primary somatosensory cortex (S1) responds dynamically to pain stimuli and plays a pivotal role in pain processing. These stimuli activate astrocytic α7-nicotinic acetylcholine receptors (α7-nAChRs), yet their contribution to pain perception remains largely unclear. This study investigates the role of astrocytic α7-nAChRs in pain information processing and perception. Astrocytic α7-nAChRs were selectively deleted by injecting rAAV5-GfaABC₁D-NLS-Cre-P2A-mCherry into the S1 region of LoxP-Chrna7 transgenic mice or by tamoxifen administration in ALDH1-CreERT2:Chrna7^loxP/loxP mice. Immunohistochemistry was used to assess the expression of α7-nAChR and gliotransmitter d-serine. Astrocytic Ca²⁺ transients were monitored in vivo using two-photon Ca²⁺ imaging following AAV5-GfaABC₁D-cytoGCaMP6f-SV40 labeling. Neuronal Ca²⁺ transients in S1 evoked by plantar electric shock were recorded via fiber photometry in freely moving mice. Nociceptive and sensory behaviors were evaluated using hot plate and von Frey tests. Statistical analyses included Welch's t-tests for normally distributed data, Mann-Whitney U tests for non-normal data, and two-way ANOVA. Results showed that conditional deletion of α7-nAChRs in astrocytes markedly reduced d-serine levels within astrocytic territories. Two-photon Ca²⁺ imaging and fiber photometry revealed that α7-nAChR deletion attenuated both astrocytic Ca²⁺ transients and footshock-evoked neuronal Ca²⁺ activity in S1. Behaviorally, thermal pain responses were significantly impaired, whereas tactile thresholds and open-field behaviors were unaffected. These findings identify astrocytic α7-nAChRs in S1 facilitate d-serine levels and enhance astrocytic activity. Their physiological function is essential for pain-related information processing within astrocyte-neuron networks and plays a critical role in regulating pain perception.

At the population level, astrocytes dynamically interact with neurons.^{1 – 3} Astrocytes sense activity within the neuronal network and respond to neural signals by generating ${\mathrm{Ca}}^{2+}$ activities in vivo.^4,5 Simultaneously, astrocyte activation or manipulation modulates neuronal network activity.^2,3 This bidirectional, interconnected communication between astrocytes and neurons is collectively termed the “astrocyte-neuronal network.”^6,7 Accumulating experimental evidence suggests that various behaviors result from coordinated activity between neurons and astrocytes within these networks.^2,8,9 Therefore, deciphering the interaction between astrocytes and neurons within astrocyte-neuronal networks is essential for comprehensively understanding the genesis and dysregulation of behavior.^2,3,10

Two-photon Ca²⁺ imaging is a widely used technique for investigating brain functions across multiple spatial scales. However, the recording of neuronal activities is affected by movement of the brain during tasks in which the animal is behaving normally. Although post-hoc image registration is the commonly used approach, the recent developments of online neuroscience experiments require real-time image processing with efficient motion correction performance, posing new challenges in neuroinformatics. We propose a fast and accurate image density feature-based motion correction method to address the problem of imaging animal during behaviors. This method is implemented by first robustly estimating and clustering the density features from two-photon images. Then, it takes advantage of the temporal correlation in imaging data to update features of consecutive imaging frames with efficient calculations. Thus, motion artifacts can be quickly and accurately corrected by matching the features and obtaining the transformation parameters for the raw images. Based on this efficient motion correction strategy, our algorithm yields promising computational efficiency on imaging datasets with scales ranging from dendritic spines to neuronal populations. Furthermore, we show that the proposed motion correction method outperforms other methods by evaluating not only computational speed but also the quality of the correction performance. Specifically, we provide a powerful tool to perform motion correction for two-photon Ca²⁺ imaging data, which may facilitate online imaging experiments in the future.