神经科学

Social behavior is highly dynamic and context-dependent, yet many commonly used rodent social assays rely on short testing periods and simplified measures of proximity or investigation. Here, we present a detailed protocol for constructing and using the immersive social interaction assay (ISIA), a behavioral paradigm designed to capture prolonged, voluntary social interactions in freely moving mice. The ISIA apparatus consists of two modified standard rodent home-cage chambers connected by a 3D-printed tube with an adjustable inner diameter. This design enables flexible experimental control: a removable restrictor can confine a head-bar-implanted focal mouse to one chamber while permitting a freely moving conspecific to traverse the full apparatus, enabling assessment of social motivation. Animals can be recorded over extended time windows with familiar or novel conspecifics, in their home territory or in novel environments, and under varying thermal or enrichment conditions, while expressing a broad repertoire of affiliative and aggressive behaviors, including huddling and aggression. The apparatus can be readily integrated with machine learning–based tracking and behavioral classification pipelines for high-throughput analysis. This protocol describes step-by-step construction, setup, and experimental implementation of the ISIA, from apparatus construction and head-bar implantation to video-based behavioral analysis, with the goal of facilitating broader adoption of ethologically relevant behavioral assays in neuroscience research.

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.

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.

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.

Structural proteomics methods allow for the proteome-wide interrogation of protein structural differences between two different conditions. Limited proteolysis mass spectrometry (LiP-MS), as originally implemented by the Picotti lab, utilizes a promiscuous protease to cleave at solvent-exposed regions of a protein to encode structural information, which is then read out with mass spectrometry proteomics. Here, we present a protocol that details experimental steps and data analysis for a LiP-MS workflow. First, tissue is homogenized under native conditions and then subjected to limited proteolysis using proteinase K (PK). The samples are prepared for mass spectrometry, and data are acquired using either data-dependent acquisition (DDA) or data-independent acquisition (DIA). Raw data is processed using FragPipe, and raw ion abundances are processed in FragPipe Limited-Proteolysis Processor (FLiPPR). Proteins with structural changes between the two conditions are identified in a proteome-wide manner.

Despite substantial progress in preclinical cannabinoid research, translational studies on cannabis use disorders (CUD) are still insufficient due to the absence of robust, validated animal models that fully recapitulate the multifactorial clinical phenotype of human CUD. The complex nature of CUD and the incomplete understanding of its underlying neurobiological mechanisms contribute to the limited availability of effective treatments. To address this gap, we developed an operant conditioning–based mouse model that enables the identification of individual vulnerability or resilience to CUD development. This highly translational model is based on the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-5) criteria for substance use disorders. The model allows the assessment of addiction-like behaviors by evaluating three behavioral domains: 1) persistence of responding during periods of cannabinoid unavailability, 2) motivation for cannabinoid seeking measured using a progressive ratio schedule, and 3) compulsivity, assessed when cannabinoid reward is paired with an aversive consequence such as a mild electric foot shock. A major strength of this paradigm is its ability to quantify two phenotypic traits proposed as predisposing factors for addiction vulnerability and two parameters related to craving. In addition, the model is specifically designed to evaluate genetic and circuit-level manipulations using chemogenetic approaches, with minor modifications required by surgical viral-vector delivery. Using this protocol, we can determine whether altering the excitability of specific neural networks promotes resilience or vulnerability to developing cannabinoid addiction. Elucidating these mechanisms is expected to facilitate the identification of novel and more effective therapeutic interventions for CUD.

Neuronal survival in vitro is usually used as a parameter to assess the effect of drug treatments or genetic manipulation in a disease condition. Easy and inexpensive protocols based on neuronal metabolism, such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), provide a global view of protective or toxic effects but do not allow for the monitoring of cell survival at the single neuronal level over time. By utilizing live imaging microscopy with a high-throughput microscope, we monitored transduced primary cortical neurons from 7–21 days in vitro (DIV) at the single neuronal level. We established a semi-automated analysis pipeline that incorporates data stratification to minimize the misleading impact of neuronal trophic effects due to plating variability; here, we provide all the necessary commands to reproduce it.

Autonomic regulation of heart and respiratory rates is essential for understanding brain–body interactions in health and disease. Preclinical cardiovascular recordings are often performed under anesthesia or via telemetry, both of which introduce physiological confounds such as stress or impaired recovery due to the need for acute or chronic implantation of sensors. Here, we present a minimally invasive protocol for simultaneous acquisition of high-quality electrocardiography and respiratory signals in awake mice. Using an in-house-modified physiological monitor in awake, head-fixed mice that were briefly habituated to experimental conditions, we ultimately enable stable, long-term physiological recordings alongside in vivo microscopy. This protocol provides a robust, low-stress method for acquiring physiological signals, enabling the simultaneous study of cardiovascular–cerebral dynamics in awake head-fixed mice, thereby enhancing the translational relevance of preclinical measurements.

Optogenetic stimulation of peripheral motor nerves is a promising technique for modulating neural activity via illumination of light-sensitive ion channels known as opsins. Stimulating muscle activity through this method offers many advantages, such as a physiological recruitment order of motor units, reduced fatigue, and target-specific stimulation, which make it a favorable option for use in many neuroscience and motor rehabilitation applications. To enable such optical stimulation, opsin expression in peripheral nerves can be achieved either with transgenic animal models or through injection of viral vectors. In this protocol, we describe a method for driving peripheral nerve opsin expression via intramuscular adeno-associated virus (AAV) injection with the goal of enhancing virus uptake by targeting injections to neuromuscular junctions with electrical stimulation. We also describe procedures for non-invasively assessing functional opsin expression over time with transdermal optical stimulation of opsin-labeled nerves and electromyography (EMG) recordings. The presence of time-locked EMG spikes 4–8 ms after each stimulation pulse demonstrates that functional opsin expression is present at a given assessment time point. Onset of functional optical sensitivity generally occurs 2–4 weeks following virus injection, and sensitivity generally peaks or plateaus between 6–10 weeks. Stimulation sequences such as light intensity, stimulation pulse width, and frequency sweeps provide further information on functional opsin expression at the testing timepoint. The methods presented here can be used for driving functional opsin expression with a standard AAV6 vector commonly used in similar experiments or as a protocol for assessing peripheral nerve opsin expression with novel viral vectors.

Microglia, the resident immune cells of the central nervous system, play a crucial role in maintaining neural homeostasis and in regulating neurodevelopment, neuroinflammation, tissue repair, and neurotoxicity. They are also key contributors to the pathogenesis of various neurodegenerative disorders, underscoring the need for in vitro models that accurately recapitulate disease-relevant conditions. Among the available isolation methods, the classical mixed glial culture shaking technique remains the most commonly employed, while alternatives such as magnetic bead separation and fluorescence-activated cell sorting (FACS) offer higher purity but are often constrained by technical complexity and cost. In this study, we refined the traditional shaking method by supplementing specific cytokines during culture to enhance microglial viability and proliferation. Our optimized protocol produced primary microglia with higher purity, greater yield, and improved viability compared with the conventional approach, thereby increasing experimental efficiency while substantially reducing time, animal usage, and overall cost.