神经科学

α-Synuclein (α-syn) aggregation has emerged as a key pathogenetic feature in several neurodegenerative disorders. The α-syn protein has various conformational strains, each with unique structural features that influence their cytotoxicity, propagation, and neuroinflammation. A post-translational modification known as O-GlcNAcylation has been found to influence the toxicity of α-syn and its propensity to aggregate. Difficulties in detecting and quantifying this modification are a major challenge to understanding its roles among the conformational forms of α-syn. We now describe a protocol for detecting O-GlcNAcylated α-syn that combines a click chemistry labeling approach and western blotting. This chemoenzymatic method involves the transfer of azido-modified galactose (GalNAz) from UDP-GalNAz to O-GlcNAcylated proteins, enabling their further functionalization with alkyne-containing polyethylene glycol of defined molecular weight. This protocol facilitates the determination of the glycosylation status of varying conformations of α-syn and their stoichiometric ratios.

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.

The genome of the nematode Caenorhabditis elegans encodes at least 160 predicted peptide precursor genes that can generate over 300 bioactive peptides, the functions of most of which remain unknown. Phenotypes resulting from deletion or transgenic expression of peptide genes are readily assayed, but genetic dissection of individual peptide activities is often confounded when a single gene encodes multiple peptides or when distinct peptides act redundantly. Here, we describe a protocol for direct microinjection of chemically synthesized peptides into individual worms. This approach permits investigation of the effects of an individual peptide while providing precise temporal control over peptide delivery.

Super-resolution imaging of synapses in intact brain tissue remains challenging because light scattering, photobleaching, and limited probe penetration, along with antigen accessibility within the densely packed postsynaptic densities (PSDs), constrain resolution and labeling efficiency. Here, we present a protocol utilizing thin brain cryosections and tau-stimulated emission depletion (STED) nanoscopy to visualize the intricate nano-architecture of excitatory synapses in situ. Slicing the brain into 6 μm sections allows for highly efficient and even penetration of probes throughout sections while ensuring that the resolution is not significantly impacted by the imaging depth of the tissue. We outline step-by-step instructions for labeling pre- and postsynaptic nano-architecture using antibodies and nanobodies, highlighting how fixative choice influences the labeling efficiency of synaptic proteins. While this protocol is compatible with both confocal and super-resolution imaging, when combined with rapid image acquisition times of tau-STED, it enables clear separation of key synaptic features in three dimensions with minimal photobleaching. Thus, this approach enables robust multiplex imaging of fluorescently labeled synaptic proteins in the brain, providing exceptional spatial resolution for visualization and quantification of synaptic nanoarchitecture in its native environment.

Organic solvent–based tissue clearing methods are widely used for whole-brain imaging but often compromise endogenous fluorescence. Existing protocols, such as iDISCO and fluorescence-preserving variants, have improved optical transparency but still present trade-offs between fluorescence retention, tissue stability, and workflow complexity. Here, we present MDISCO, a modified iDISCO-based clearing protocol designed to enhance preservation of endogenous fluorescence while maintaining high transparency and stable tissue morphology. MDISCO is directly compared with FDISCO+, an established fluorescence-preserving protocol, for the preservation of endogenous tdTomato and YFP. Performance across clearing steps is evaluated by measuring brain weight, anteroposterior and mediolateral dimensions, and optical transparency before and after solvent clearing and refractive index matching. Fluorescence preservation is assessed using whole-brain light-sheet microscopy with standardized imaging parameters to enable direct comparison. This protocol provides an accessible and high-throughput, reproducible workflow for solvent-based clearing with robust endogenous fluorescence preservation, offering clear advantages for whole-brain 3D imaging of genetically encoded fluorescent reporters.

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.

The morphology of single-neuron axonal projections is critical for deciphering neural circuitry and information flow in the brain. Yet, manually reconstructing these complex, long-range projections from high-throughput whole-brain imaging data remains an exceptionally labor-intensive and time-consuming task. Here, we developed a points assignment-based method for axonal reconstruction, named PointTree. PointTree enables the precise identification of the individual axons from densely packed axonal population using a minimal information flow tree model to suppress the snowball effect of reconstruction errors. In this protocol, we have elaborated on how to configure the required environment for PointTree software, prepare suitable data for it, and run the software. This protocol can assist neuroscience researchers in more easily and rapidly obtaining the reconstruction results of neuronal axons.

Amphibian retinas contain “green” rods, which are rod-shaped photoreceptors with a cone-type visual pigment. These rods are considered a potentially transitional photoreceptor type, but their phototransduction cascade’s molecular composition has remained uncertain. Here, we present a streamlined electrophysiology-molecular workflow that enables the rapid spectral identification, physical capture, and targeted single-cell reverse transcription-polymerase chain reaction (RT-PCR) of individual amphibian photoreceptors. After suction-pipette spectral screening under alternating red and green illumination, electrophysiologically identified cells are isolated and processed directly for reverse transcription and PCR. Coupling real-time functional phenotyping with sensitive molecular profiling provides a practical tool for resolving photoreceptor molecular heterogeneity and investigating evolutionary transitions between rod and cone phenotypes.

This protocol describes a reproducible workflow for modeling in vitro impact-induced traumatic brain injury (TBI) using a mechanical stretch system applied to differentiated SH-SY5Y human neuroblastoma cells cultured on polydimethylsiloxane (PDMS) substrates. The protocol integrates three primary components: (1) fabrication and surface modification of deformable PDMS chambers to support cellular adhesion, (2) partial differentiation of SH-SY5Y cells using retinoic acid, and (3) induction of controlled mechanical strain to simulate mild to moderate TBI. The stretch-induced injury model enables quantitative assessment of cellular viability and recovery following mechanical insult. This approach provides a versatile platform for studying cellular and molecular mechanisms of TBI, screening neuroprotective compounds, and exploring mechanobiological responses in neural cells under controlled strain magnitudes and rates.

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.