Cell Biology

Since the discovery that astrocytes are characterized by Ca²⁺-based excitability, investigating the function of these glial cells within the brain requires Ca²⁺ imaging approaches. The technical evolution from chemical fluorescent Ca²⁺ probes with low cellular specificity to genetically encoded indicators (GECIs) has enabled detailed analysis of the spatial and temporal features of intracellular Ca²⁺ signal. Different imaging methodologies allow the extraction of distinct information on calcium signals in astrocytes from brain slices, with resolution ranging from cell populations to single cells up to subcellular domains.

Here, we describe 2-photon laser scanning microscopy (2PLSM) Ca²⁺ imaging in astrocytes from the somatosensory cortex (SSCx) of adult mice in ex vivo acute cortical slices, performed using two genetically encoded Ca²⁺ indicators, i.e., cytosolic GCaMP6f and endoplasmic reticulum-targeted G-CEPIA1er. The main advantage of the 2PLSM technique, compared to single-photon microscopy, is the possibility to go deeper in the tissue while avoiding photodamage, by limiting laser excitation to a single focal plane. The fluorescent signal of the indicator is analyzed offline in different compartments—soma, proximal processes, and microdomains—for GCaMP6f experiments and in the perinuclear, somatic area for G-CEPIA1er. The analysis of Ca²⁺ signal from different compartments, although not providing a value of absolute concentration, allows a critical comparison of the degree of astrocyte activation between different experimental conditions or mouse models. Moreover, the analysis of G-CEPIA1er signal, which reveals metabotropic receptor activation as a dynamic decrease in free Ca²⁺ in the endoplasmic reticulum (ER), can provide information on possible alterations in this critical second messenger pathway in astrocytes, including, for example, steady-state ER Ca²⁺ levels and kinetics of Ca²⁺ release.

In vivo two-photon imaging of the mouse brain is essential for understanding brain function in relation to neural structure; however, its application is limited by the size and mechanical stability of conventional cranial windows. Here, we present the procedure of a large-scale cranial window technique based on the nanosheet incorporated into light-curable resin (NIRE) method. This approach utilizes a biocompatible polyethylene-oxide-coated CYTOP (PEO-CYTOP) nanosheet combined with light-curable resin, allowing the window to conform to the brain’s curved surface. The protocol enables long-term, high-resolution, and multiscale imaging—from subcellular structures to large neuronal populations—in awake mice over several months.

Research into nervous system injuries and regeneration has emerged as a crucial field of study. In many cases such as trauma or stroke, both axons and dendrites are equally damaged; however, studying injury and repair mechanisms in both neurite processes (axons and dendrites) of the same neuron has been challenging. Additionally, correlating the behavioral aspects of neuronal injury with anatomical regeneration is important for a better understanding of the functional rewiring process. Here, we describe protocols for injuring the dendrites and the axon of the PVD neuron of C. elegans using a two-photon infrared (IR) femtosecond laser system, and subsequent imaging of injured neurites during the course of regeneration. Additionally, we describe the protocols for the behavioral study concerning the PVD neuron and their analysis, which can offer valuable insights. These assays can be implemented to assess the function of the pathways that play specific roles in dendrite vs. axon regeneration.

The reduction in intracellular neuronal chloride concentration is a crucial event during neurodevelopment that shifts GABAergic signaling from depolarizing to hyperpolarizing. Alterations in chloride homeostasis are implicated in numerous neurodevelopmental disorders, including autism spectrum disorder (ASD). Recent advancements in biosensor technology allow the simultaneous determination of intracellular chloride concentration of multiple neurons. Here, we describe an optimized protocol for the use of the ratiometric chloride sensor SuperClomeleon (SClm) in organotypic hippocampal slices. We record chloride levels as fluorescence responses of the SClm sensor using two-photon microscopy. We discuss how the SClm sensor can be effectively delivered to specific cell types using virus-mediated transduction and describe the calibration procedure to determine the chloride concentration from SClm sensor responses.

The inferior colliculus (IC) is an important processing center in the auditory system, which also receives non-auditory sensory input. The IC consists of several subnuclei whose functional role in (non-) auditory processing and plastic response properties are best approached by studying awake animals, preferably in a longitudinal fashion. The increasing use of mice in auditory research, the availability of genetic models, and the superficial location of the IC in the mouse have made it an attractive species for studying IC function. Here, we describe a protocol for exposing the mouse IC for up to a few weeks for in vivo imaging or electrophysiology in a stable manner. This method allows for a broader sampling of the IC while maintaining the brain surface in good quality and without reopening the craniotomy. Moreover, as it is adaptable for both electrophysiological recordings of the entire IC and imaging of the dorsal IC surface, it can be applied to answer a multitude of questions.

Key features

• A surgical protocol for long-term physiological recordings from the same or separate neuronal populations in the inferior colliculus.

• Optimized for awake in vivo experiments in the house mouse (Mus musculus).

Over the past years, research has made impressive breakthroughs towards the development and implementation of 3D cell models for a wide range of applications, such as drug development and testing, organogenesis, cancer biology, and personalized medicine. Opposed to 2D cell monolayer culture systems, advanced 3D cell models better represent the in vivo physiology. However, for these models to deliver scientific insights, appropriate investigation techniques are required. Despite the potential of fluorescence microscopy to visualize these models with high spatial resolution, sample preparation and imaging assays are not straightforward. Here, we provide different protocols of sample preparation for fluorescence imaging, for both matrix-embedded and matrix-free models (e.g., organoids and spheroids, respectively). Additionally, we provide detailed guidelines for imaging 3D cell models via confocal multi-photon fluorescence microscopy. We show that using these protocols, images of 3D cell culture systems can be obtained with sub-cellular resolution.

Graphical abstract:

Changes in intracellular calcium drive exocrine cell activity. In the salivary gland, acetylcholine released from parasympathetic neurons mobilizes endoplasmic reticulum calcium stores in acinar cells, which consequently initiates saliva secretion. However, our understanding of the signaling cascade is mainly based on ex vivo studies performed in enzymatically isolated cells. The dissociation process likely disrupts the extracellular matrix, removes neurons as the source of signal input, and disturbs the integrity of tight and gap junctional acinar connections. These alterations may affect the spatiotemporal properties of calcium signaling events. In vivo observations of calcium signals, where tissue organization is intact, are therefore important to establish the characteristics of physiological calcium signals that are crucial for the stimulation of fluid secretion. Here, we present a detailed protocol for in vivo imaging of calcium signaling events, following nervous stimulation by multi-photon microscopy in mouse salivary gland acinar cells, expressing the genetically encoded calcium indicator GCamp6F.

Cytokinesis occurs at the final step of cell division and leads to the separation of daughter cells. It requires assembly and constriction of the actomyosin contractile ring. The phases of assembly and constriction of the contractile ring show an increase in tension in the actomyosin complex. The measurement of tension in the contractile ring is of interest to probe the mechanics of contractile ring formation. Drosophila cellularization is a powerful genetic model system to study the mechanisms regulating actomyosin contractility during contractile ring constriction. Cellularization occurs in the interphase of syncytial cycle 14, where the plasma membrane extends around individual nuclei and forms complete cells with the help of a contractile ring at the bottom. The contractile ring forms at the furrow tip during the extension around individual nuclei and its assembly requires the coordinated action of cytoskeletal and plasma membrane-associated proteins. Laser ablation of the contractile ring enables the measurement of the contractility of the actomyosin network during cytokinesis. This protocol outlines the method used for estimating the contractility at the actomyosin ring during cellularization by laser ablation, in both control and mutant embryos for a Rho guanosine triphosphatase activating protein (RhoGAP) containing protein called GRAF (GTPase regulator associated with focal adhesion kinase-1). Physical cutting of the contractile ring by a two-photon laser at 800 nm leads to the displacement of the actomyosin ring edges, at a rate dependent upon the tension. This can be carried out at distinct steps of the contractile ring assembly during furrow extension in cellularization. Quantification of the extent of displacement and recoil velocity of movement of the edges at different stages of cellularization provides a quantitative measure of contractility in the system. This protocol describes the experimental procedure containing the preparation of live embryos, optimization of laser power, acquisition settings, and post-acquisition analysis of actomyosin contractility during Drosophila cellularization.

T follicular helper (Tfh) cells regulate B cell selection for entry into the germinal center (GC) reaction or for differentiation into antibody forming cells. This process takes place at the border between the T and B zones in lymphoid organs and involves physical contacts between T and B cells. During these interactions, T cells endow the B cells with selection signals that promote GC seeding or plasmablast differentiation based on their B cell receptor affinity. In Peyer’s patches (PPs), T cells promote B cell colonization of the subepithelial dome (SED) without effective affinity-based clonal selection. To specifically characterize the T cell population that resides within the SED niche, we performed ex vivo photoactivation of the SED compartment followed by flow cytometry analysis of the labeled cells, as described in this protocol. This technique integrates both spatial and cellular information in studies of immunological niches and can be adapted to various experimental systems.

Photothrombosis of blood vessels refers to the activation of a circulating photosensitive dye with a green light to induce clotting in vivo (Watson et al., 1985). Previous studies have described how a focused green laser could be used to noninvasively occlude pial arterioles and venules at the brain surface (Schaffer et al., 2006; Nishimura et al., 2007; Shih et al., 2013). Here we show that small regions of the capillary bed can similarly be occluded to study the ischemic response within the capillary system of the mouse cerebral cortex. The advantage of this approach is that the ischemic zone is restricted to a diameter of approximately 150-250 μm. This permits higher quality two-photon imaging of degenerative processes that would be otherwise difficult to visualize with models of large-scale stroke, due to excessive photon scattering. A consequence of capillary occlusion is leakage of the blood-brain barrier (BBB). Here, through the use of two-photon imaging data sets, we show how to quantify capillary leakage by determining the spatial extent and localization of intravenous dye extravasation.