Neuroscience

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.

Adaptive behaviors shaped by prior experience are essential for increasing animal survival. Aversive experiences play a pivotal role in memory formation and in updating subsequent learning rules. While the negative value of aversive signals, which are both necessary and sufficient to drive a conditioned response, is considered to be innately specified, it can also be subject to experience-dependent scaling. Previous reports demonstrated synaptic potentiation in nociceptive pathways following robust aversive learning. However, the neuronal basis of experience-dependent value updating remains largely unknown. Recently, we demonstrated that long-term potentiation (LTP) in the parabrachial-central amygdala (PB-CeA) pathway, an important circuit involved in pain processing and aversive learning, enhances the negative value and thereby updates future learning rules. Here, we present a protocol that combines behavioral analysis using pathway-specific optogenetic induction of in vivo LTP with mathematical modeling to examine value modification using Bayesian inference of the unconditioned stimulus value using the Rescorla–Wagner model. This protocol enables investigation of the mechanisms underlying experience-dependent value modulation and learning-rule changes in mice. Potentially, this protocol may provide a framework for understanding learning rules across a wide range of species and for the development of treatments for stress-related disorders.

Three-dimensional immunohistochemistry (3D-IHC) shows the organization of molecular assemblies in the context of tissue architecture. Deep and rapid antibody penetration into 3D tissues and highly sensitive detection are crucial for high-throughput analysis of 3D-IHC imaging. Here, we provide a detailed protocol for a nanobody (nAb)-based 3D-IHC technique, namely POD-nAb/FT-GO 3D-IHC, for high-speed and high-sensitivity detection of targets within 1-mm-thick mouse brain tissues. Peroxidase-fused nAb (POD-nAb) is a genetically encoded recombinant antibody, which consists of a camelid nAb and a variant of horseradish peroxidase, and fluorochromized tyramide-glucose oxidase (FT-GO) is a fluorescent tyramide signal amplification (TSA) system. POD-nAb/FT-GO 3D-IHC incorporates three main components: 1) tissue permeabilization, 2) POD-nAb binding, and 3) 3D-TSA reaction with FT-GO. POD-nAbs enhance signal penetration depth and allow for highly sensitive detection when combined with FT-GO signal amplification. By using the 3D-IHC protocol provided herein, we can visualize target molecules in mouse brain tissues of 1-mm thickness with drastic signal enhancement within three days. This protocol for POD-nAb/FT-GO 3D-IHC could facilitate structural and molecular interrogation of 3D tissues.

Animal and human stem cell–derived three-dimensional models to study physio-pathological brain functioning are becoming a gold standard for in vitro electrophysiology, as they enable the recapitulation of complex network properties by accounting for spatial architectural features that better reflect in vivo conditions than simpler 2D models. Standard planar multielectrode arrays (MEAs), typically providing tens of recording electrodes, are commonly used to record activity from 2D neuronal cultures. However, when adapted for use with 3D models, planar 2D MEAs showed limited effectiveness. The main issues are limited specimen adhesion to the chip, a low number of sensing elements, inability to retrieve signals from within the tissue, and reduced perfusion and vitality of the tissue in contact with sensors. To overcome these limitations, a new generation of microchip-based 3D high-density MEAs (3D HD-MEA) has been developed and validated in recent years. This technological advancement has improved the sensing capabilities and the vitality of 3D models, providing a tool tailored to maximize their potential. Here, we present an optimized protocol for neural network activity recordings in 3D models (including acute slices, brain spheroids, and organoids) from various brain regions using 3D HD-MEAs. First, we summarize the critical steps for 1) obtaining viable acute slices from the mouse cerebellum, cortico-hippocampal circuit, and prefrontal cortex, 2) establishing efficient coupling of the slices with the chip, and 3) performing recordings and analyses. We then describe the main procedures required to obtain human and animal brain spheroids and neural organoids, as well as standardized routines to perform effective recordings and analyses. For each section, we highlight the crucial steps, identify tips for specific applications, and propose troubleshooting procedures. For example, the same type of preparation (e.g., acute slices) requires different adjustments when working with different brain areas. The specific information provided here is intended to assist researchers in their daily efforts to obtain efficient and reproducible functional recordings from 3D models by using the cutting-edge technique of 3D HD-MEA.

Postnatal mouse retinal vascular development is a widely used model for studying retinal vascular diseases and evaluating candidate therapies. This is particularly relevant for inherited disorders such as familial exudative vitreoretinopathy (FEVR), in which impaired vascular growth and organization are central to disease pathogenesis. Numerous approaches have been used to assess retinal vasculature in mouse flat mounts, ranging from qualitative descriptions to limited quantitative measurements of vascular growth. However, phenotypic variability across genetic models, including different models of FEVR, complicates comparisons and underscores the need for standardized, comprehensive multi-parameter analyses that are suitable for rapid and cost-effective screening studies. We describe a standardized morphometric protocol using ImageJ software to quantitatively analyze mouse retinal vasculature in a reproducible manner. The protocol begins with measurement of areas of vascular disorganization (meshes) as well as total vascular and retinal area. Two defined regions in the peripheral and midperipheral retina are then selected to quantify cell clusters, followed by image processing, binarization, and skeletonization. From these processed images, vascular density, branch number, branch length and thickness, junction number, triple points, and box-counting fractal dimension and lacunarity are quantified. Overall, this protocol provides a rapid, cost-effective, and standardized framework for quantifying retinal vascular phenotypes across diverse mouse models. By capturing multiple structural features and accommodating phenotypic variability, it is well-suited for comparative studies and therapeutic screening in retinal vascular disease.