Neuroscience

Expressing large DNA constructs in the native three-dimensional brain microenvironment remains technically challenging. Although viral vectors provide high transduction efficiency and cell-type selectivity, their genetic payload capacity is limited. Various non-viral approaches have been used in brain tissue, but they may compromise tissue viability or require specialised equipment, such as biolistic delivery or electroporation. We present an adapted protocol for delivering the large DNA vector encoding the optical PIEZO1 sensor GenEPi into brain tissue to enable sensor expression in pyramidal neurons. By applying DNA–Lipofectamine liposomes directly to the slice surface, we achieved efficient, minimally invasive transfection of pyramidal neurons in the CA1 and CA3 regions of organotypic hippocampal slices. PIEZO1 sensor expression was detectable as early as 7 days after transfection, increased with longer tissue maintenance, and was sustained for 3–4 weeks in vitro. This protocol describes a cost-effective, non-invasive approach that preserves cell viability and enables investigation of PIEZO1-mediated mechanotransduction in a native brain microenvironment.

Progressive neurodegeneration linked to the accumulation of misfolded proteins is a hallmark of several neurodegenerative disorders, including Parkinson’s disease, Huntington’s disease, and Alzheimer’s disease. Dysfunction in the protein homeostasis machinery correlates with pathology. The chaperone protein DNAJB6 is expressed in neurons and oligodendrocytes and has been shown to play a key role in preventing amyloid aggregation by binding to amyloidogenic proteins and facilitating their refolding or degradation, in cooperation with other chaperones. Here, we describe a simple and feasible assay that enables high-throughput screening for DNAJB6 activity in a plate reader format. We use genetically engineered HEK293 cells that stably express DNAJB6 fused to either CFP or YFP. These cells can be plated into multi-well plates, and the fluorescence resonance energy transfer (FRET) signal can be measured for analysis of DNAJB6 dimerization, which is linked to DNAJB6 activity. The protocol can be used for drug screening and to identify compounds that increase DNAJB6 dimerization, and can serve as a starting point for finding new medicines that act through modulating DNAJB6 activity.

Retinopathy of prematurity (ROP), a retinovascular disease, is a leading cause of childhood blindness worldwide. Given the constraints of studying molecular mechanisms in preterm infants, reproducible animal models are important to understand ROP pathophysiology. Mouse and rat oxygen-induced retinopathy (OIR) models are the most commonly used and recapitulate key vascular features seen in ROP. However, these models are susceptible to inherent variability that limits reproducibility, including inter-litter variability, consistency of oxygen delivery across experiments, retinal dissection technique, and immunohistochemistry. Here, we describe a comprehensive protocol for performing the most common mouse and rat OIR models, and procedures such as eye enucleation, retinal dissection and flat mounting, isolectin GS-IB4 staining, whole retina stitched fluorescence imaging from Z-stacks, and quantification of vascular features. This protocol provides important materials and procedural details to increase the reproducibility of the mouse and rat OIR models.

Phosphatase and tensin homolog-induced kinase 1 (PINK1) is a serine/threonine kinase that plays a key role in mitophagy initiation. Loss-of-function autosomal recessive mutations in PINK1 cause early onset Parkinson’s disease (EOPD). Current approaches for studying PINK1 function depend on bulk techniques that can only provide snapshots of activity and could miss the dynamics and cell-to-cell heterogeneity of PINK1 activity or provide an indirect readout of PINK1 activity. Here, we present a protocol using our newly developed phase separation–based PINK1 biosensor (PINK1-SPARK) to observe real-time activity of endogenous PINK1 in single cells. Following transfection of live cells with PINK1-SPARK, cells are treated with mitochondrial depolarizing agents and visualized using widefield or confocal fluorescence microscopy, either following the same cells over time for time-lapse imaging of PINK1 activity or end-point measurements. Thus, PINK1-SPARK is a new tool that enables the measurement of PINK1 activity in single live cells, allowing for further elucidation of the role of PINK1 in mitophagy and cell function.

Persistent neural activity underlies fundamental brain functions such as memory, decision-making, and emotion. Despite its importance, experimental paradigms that enable quantitative analysis of persistent behavioral responses remain limited. Here, we describe a protocol to induce and measure a persistent locomotor response by applying a brief alternating current (AC) electric stimulus to the nematode Caenorhabditis elegans. This method reliably evokes a prolonged increase in locomotion speed that persists for minutes after stimulus termination and can be quantified by video tracking. Because C. elegans has a fully mapped connectome and is amenable to genetic and neurophysiological manipulation, this protocol provides a useful platform for dissecting the molecular and neural mechanisms underlying persistent behavioral responses. Electrically induced persistent locomotion serves as a simple, robust, and quantifiable behavioral readout for studying the regulation of neural persistence in vivo.

Standardized laboratory assays are essential for generating reproducible and comparable data in toxicology. Although acute contact and oral toxicity tests are widely applied in pesticide risk assessment, these approaches have rarely been adapted for social vespids. Vespa velutina nigrithorax, an invasive hornet in Europe and East Asia, is commonly managed through chemical control, yet treatment efficacy may vary depending on the route of exposure and other biological factors. This protocol describes a standardized method to assess acute contact and oral toxicity of chemical compounds in adult V. v. nigrithorax workers under controlled laboratory conditions. Hornets are collected in the field, individually housed, and exposed either to topical applications on the thorax or to spiked food sources. Mortality is monitored over 48–96 h and analyzed using appropriate statistical approaches to estimate lethal endpoints. This protocol enables comparison among compounds and exposure routes and provides a practical framework for toxicity screening in hornets.

Whole-mount techniques are widely used in medical and biological research to analyze protein expression and tissue organization in intact specimens. Traditional approaches for protein localization include section-based immunohistochemistry and in situ hybridization; however, these methods can be limited by tissue disruption and loss of spatial context. Whole-mount protocols generally involve tissue fixation, permeabilization, and staining with specific probes, but their effectiveness varies depending on the antigen–antibody combination and the specimen type. Consequently, no universal protocol is suitable for all experimental conditions. This protocol presents a detailed whole-mount immunostaining protocol for evaluating tyrosine hydroxylase (TH) expression, a key marker of dopaminergic neurons, in zebrafish (Danio rerio) larvae. The procedure outlines critical steps from sample preparation to staining optimization to ensure reproducible and specific signal detection. This approach enables accurate visualization and analysis of dopaminergic neuron distribution in intact larvae. The protocol offers a reliable and adaptable approach that preserves tissue integrity, enables three-dimensional visualization, and is particularly well-suited for developmental and neurobiological studies using zebrafish larvae.

Real-time measurement of blood flow and nanocarrier transport in the cerebral microvasculature is crucial for understanding neurovascular physiology and nanocarrier-based drug delivery. Existing techniques lack the ability to measure blood flow rates in individual vessels with high spatial and temporal resolution in real time. Two-photon fluorescence correlation spectroscopy (2P-FCS) provides a powerful approach for monitoring tracer molecules within a small confocal observation volume. This enables the simultaneous determination of particle number and flow dynamics in vivo. Here, we present a detailed protocol for in vivo 2P-FCS measurements in the mouse cerebral microvasculature. The protocol includes preparation of the cranial window, delivery of fluorescent dextran tracers for vascular visualization, and FCS measurements. It also includes two-photon imaging of the cerebrovascular network and acquisition and analysis of fluorescence correlation data. The protocol describes calibration of the confocal volume diameter and optimization of two-photon excitation parameters. This workflow enables real-time measurement of tracer concentration and flow velocity in individual cerebral microvessels with high spatial and temporal resolution. The method can be adapted to study blood flow dynamics, nanoparticle transport, and microvascular physiology in a variety of in vivo imaging systems equipped with multiphoton microscopy and FCS capabilities.

Functional imaging of neural structures at the base of the cranium, including the trigeminal ganglion (TG), is technically challenging due to limited optical access. The TG—the largest sensory ganglion in the head—houses primary afferent neurons that relay information from the teeth, oral cavity, and face, yet investigation of somatosensory processing at the population level has remained limited. Here, we present a surgical procedure for an optical-window preparation that enables direct optical access to the TG. The ganglion is exposed by a large temporal craniotomy with removal of overlying tissue, and a glass cuboid is then placed in direct contact with the TG to suppress motion while maintaining the cranial cavity as a closed compartment without continuous perfusion. This preparation allows reliable visualization and recording of individual TG neurons during controlled stimulation of diverse facial and intraoral sites. Our approach provides a practical platform to map peripheral sensory representations within the TG and to investigate mechanisms underlying dental sensation, orofacial pain, and trigeminal circuit function.

Understanding gene expression within defined neuronal populations is essential for dissecting the cellular and molecular diversity of the brain. mRNA assays provide a direct readout of gene expression, capturing transcriptional changes that may precede or occur independently of protein abundance, whereas protein assays reflect the cumulative effects of translation, modification, and degradation. Moreover, in histological analysis, immunohistochemical protein detection results in visually diffuse labeling, which makes it difficult to quantitatively assess levels and locations of expression at high resolution. Here, we present a protocol that allows for mRNA detection in single neuronal cell types with a high degree of sensitivity and anatomical resolution. This protocol combines fluorescent in situ hybridization (FISH) with immunohistochemistry (IHC) on the same tissue section. Briefly, FISH is carried out by ACDBio RNAscope^® fluorescent in situ hybridization technology, which involves processing the tissue sections, followed by signal amplification. This involves target retrieval, probe hybridization, and signal enhancement. Then, the tissue section is processed for IHC, which involves blocking nonspecific sites and incubation with primary antibodies, followed by development of a fluorescent signal with secondary antibodies. Typically, visual mRNA detection with FISH can be seen as individual puncta, whereas targeting the protein with an antibody results in filled cells or processes. The variation in staining pattern allows for the quantification of distinct mRNA transcripts within different neuronal populations, which renders co-localization analyses easy and efficient.