神经科学


分类

现刊
0 Q&A 96 Views Jul 5, 2025

We recently developed an approach for cell type–specific CRISPR/Cas9 editing and transgene expression using a single viral vector. Here, we present a protocol describing how to design and generate plasmids and adeno-associated viruses (AAVs) compatible with this single-vector gene editing approach. This protocol has four components: (1) guide RNA (gRNA) design to target specific genes of interest, (2) ligation and cloning of CRISPR-competent AAV vectors, (3) production of vector-containing AAVs, and (4) viral titer quantification. The resultant vectors are compatible for use with mouse lines expressing the Cas9 protein from Streptococcus pyogenes (SpCas9) and Cre recombinase to enable selective co-expression of standard neuroscience tools in edited cells. This protocol can produce AAVs of any serotype, and the resulting AAVs can be used in the central and peripheral nervous systems. This flexible approach could help identify and test the function of novel genes affecting synaptic transmission, circuit activity, or morphology with a single viral injection.

0 Q&A 67 Views Jul 5, 2025

The fatal motor neuron (MN) disease amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of the phrenic MNs (phMNs) controlling the activity of the diaphragm, leading to death by respiratory failure. Human experimental models to study phMNs are lacking, hindering the understanding of the mechanisms of phMN degeneration in ALS. Here, we describe a protocol to derive phrenic-like MNs from human induced pluripotent stem cells (hiPSC-phMNs) within 30 days. During spinal cord development, phMNs emerge from specific MN progenitors located in the dorsalmost MN progenitor (pMN) domain at cervical levels, under the control of a ventral-to-dorsal gradient of Sonic hedgehog (SHH) signaling and a rostro-caudal gradient of retinoic acid (RA). The method presented here uses optimized concentrations of RA and the SHH agonist purmorphamine, followed by fluorescence-activated cell sorting (FACS) of the resulting MN progenitor cells (MNPCs) based on a cell-surface protein (IGDCC3) enriched in hiPSC-phMNs. The resulting cultures are highly enriched in MNs expressing typical phMN markers. This protocol enables the generation of hiPSC-phMNs and is highly reproducible using several hiPSC lines, offering a disease-relevant system to study mechanisms of respiratory MN dysfunction. While the protocol has been validated in the context of ALS research, it can be adopted to study human phrenic MNs in other research fields where these neurons are of interest.

0 Q&A 53 Views Jul 5, 2025

Over the lifespan of an individual, brain function requires adjustments in response to environmental changes and learning experiences. During early development, neurons overproduce neurite branches, and neuronal pruning removes the unnecessary neurite branches to make a more accurate neural circuit. Drosophila motoneurons prune their intermediate axon bundles rather than the terminal neuromuscular junction (NMJ) by degeneration, which provides a unique advantage for studying axon pruning. The pruning process of motor axon bundles can be directly analyzed by real-time imaging, and this protocol provides a straightforward method for monitoring the developmental process of Drosophila motor neurons using live cell imaging.

0 Q&A 121 Views Jul 5, 2025

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.

0 Q&A 79 Views Jul 5, 2025

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.

0 Q&A 63 Views Jul 5, 2025

Zika virus (ZIKV), an arthropod-borne orthoflavivirus, has emerged as a global health concern due to its ability to cause severe fetal neurological disorders, leading to the congenital Zika syndrome (CZS) in neonates. Vertical transmission during pregnancy can alter neural progenitor cell (NPC) proliferation and differentiation and induce apoptosis, leading to microcephaly and other neurodevelopmental abnormalities. While mammalian models have been used to study the impact of ZIKV on NPC behavior, limitations such as high costs, dedicated time, and ethical constraints have fostered the exploration of alternative systems. The zebrafish embryo constitutes an advantageous in vivo model for studying ZIKV neuropathogenesis. Indeed, ZIKV infection phenocopies several features of the CZS while sharing a conserved neuroanatomical layout and offering genetic plasticity and unique accessibility to the infected brain compared to mammals. Here, we describe a protocol for characterizing ZIKV-induced defects of NPCs in this zebrafish model, relying on whole animal flow cytometry.

往期刊物
0 Q&A 139 Views Jun 20, 2025

Active sampling, such as respiration, is known to play a major role in modulating how sensory information is perceived and encoded in the field of olfaction. Hence, monitoring respiration is crucial for understanding olfactory-guided behavior and physiology. Several methods used to measure respiration, such as infrared cameras, piezoelectric sensors, video monitoring, temperature probes, intubation, and intranasal cannula, require the animal or at least its head to be fixed. However, telemetry-based sensors can be used wirelessly, allowing animals to move freely. Here, we describe the surgical protocol to implant a telemetry pressure sensor in the internal jugular vein to detect changes in thoracic pressure. The sensor can thus help in monitoring respiration by transmitting the signal wirelessly. We describe a way of inserting the probe into the right jugular vein aseptically while housing the transmitter underneath the skin on the back of the animal. Next, based on the optimal spot for the best signal, we secure the position of the probe and suture the skin. The animal then undergoes regular post-operative care with painkillers and soft diets for up to a week. The method offers two main advantages; first, it uses a strategy similar to the jugular vein catheterization, which is widely established in rodents. Second, it minimizes the need for extensive post-operative care, including not having to shift to a liquid diet post-recovery. This makes the animals fit for most behavioral experiments requiring water or food restrictions.

0 Q&A 140 Views Jun 20, 2025

Primary oligodendrocyte cultures are a crucial driving force for in vitro research on oligodendrocytes (OLs) and myelin. Various methods are available to obtain oligodendrocyte lineage cells, primarily from neonatal rodent brains or human induced pluripotent stem cells (iPSCs). In this protocol, we describe a step-by-step procedure for detaching and cryopreserving primary rat oligodendrocyte progenitor cells (OPCs), followed by the thawing, proliferation, and differentiation of the cryopreserved OPCs. After freezing in a serum-free cryopreservation medium, the OPCs can be preserved at -80 °C for up to two months without notable changes in viability, proliferation, or differentiation into mature OLs. Cryopreserved OPCs can be differentiated into mature OLs with robust myelin processes and the capacity to wrap around neuron-mimicking structures. Combined with the author’s method for primary OL culture, which allows for bulk production of OPCs, OPC cryopreservation may substantially improve the efficiency of in vitro OL research.

0 Q&A 199 Views Jun 20, 2025

The neuromuscular junction (NMJ) is critical for muscle function, and its dysfunction underlies conditions such as sarcopenia and motor neuron diseases. Current protocols for assessing NMJ function often lack standardized stimulation parameters, limiting reproducibility. This study presents an optimized ex vivo method to evaluate skeletal muscle and NMJ function using the Aurora Scientific system, incorporating validated stimulation protocols for both nerve and muscle to ensure consistency. Key steps include tissue preparation in a low-calcium, high-magnesium solution to preserve NMJ integrity, determination of optimal muscle length, and sequential stimulation protocols to quantify neurotransmission failure and intratetanic fatigue. By integrating rigorous standardization, this approach enhances reproducibility and precision, providing a robust framework for investigating NMJ pathophysiology in aging and disease models.

0 Q&A 170 Views Jun 20, 2025

Human brain development relies on a finely tuned balance between the proliferation and differentiation of neural progenitor cells, followed by the migration, differentiation, and connectivity of post-mitotic neurons with region-specific identities. These processes are orchestrated by gradients of morphogens, such as FGF8. Disruption of this developmental balance can lead to brain malformations, which underlie a range of complex neurodevelopmental disorders, including epilepsy, autism, and intellectual disabilities. Studying the early stages of human brain development, whether under normal or pathological conditions, remains challenging due to ethical and technical limitations inherent to working with human fetal tissue. Recently, human brain organoids have emerged as a powerful in vitro alternative, allowing researchers to model key aspects of early brain development while circumventing many of these constraints. Unlike traditional 2D cultures, where neural progenitors and neurons are grown on flat surfaces, 3D organoids form floating self-organizing aggregates that better replicate the cellular diversity and tissue architecture of the developing brain. However, 3D organoid protocols often suffer from significant variability between batches and individual organoids. Furthermore, few existing protocols directly manipulate key morphogen signaling pathways or provide detailed analyses of the resulting effects on regional brain patterning.


To address these limitations, we developed a hybrid 2D/3D approach for the rapid and efficient induction of telencephalic organoids that recapitulate major steps of anterior brain development. Starting from human induced pluripotent stem cells (hiPSCs), our protocol begins with 2D neural induction using small-molecule inhibitors to achieve fast and homogenous production of neural progenitors (NPs). After dissociation, NPs are reaggregated in Matrigel droplets and cultured in spinning mini-bioreactors, where they self-organize into neural rosettes and neuroepithelial structures, surrounded by differentiating neurons. Activation of the FGF signaling pathway through the controlled addition of FGF8 to the culture medium will modulate regional identity within developing organoids, leading to the formation of distinct co-developing domains within a single organoid. Our protocol combines the speed and reproducibility of 2D induction with the structural and cellular complexity of 3D telencephalic organoids. The ability to manipulate signaling pathways provides an additional opportunity to further increase system complexity, enabling the simultaneous development of multiple distinct brain regions within a single organoid. This versatile system facilitates the study of key cellular and molecular mechanisms driving early human brain development across both telencephalic and non-telencephalic areas.

0 Q&A 387 Views Jun 5, 2025

AMPA-type receptors are transported large distances to support synaptic plasticity at distal dendritic locations. Studying the motion of AMPA receptor+ vesicles can improve our understanding of the mechanisms that underlie learning and memory. Nevertheless, technical challenges that prevent the visualization of AMPA receptor+ vesicles limit our ability to study how these vesicles are trafficked. Existing methods rely on the overexpression of fluorescent protein-tagged AMPA receptors from plasmids, resulting in a saturated signal that obscures vesicles. Photobleaching must be applied to detect individual AMPA receptor+ vesicles, which may eliminate important vesicle populations from analysis. Here, we present a protocol to study AMPA receptor+ vesicles that addresses these challenges by 1) tagging AMPA receptors expressed from native loci with HaloTag and 2) employing a block-and-chase strategy with Janelia Fluor-conjugated HaloTag ligand to achieve sparse AMPA receptor labeling that obviates the need for photobleaching. After timelapse imaging is performed, AMPA receptor+ vesicles can be identified during image analysis, and their motion can be characterized using a single-particle tracking pipeline.

0 Q&A 228 Views Jun 5, 2025

Long-term depression (LTD), a key form of synaptic plasticity, is typically induced through regulated Ca2+ entry via NMDA receptors and achieved by prolonged (up to hundreds of seconds) low-frequency presynaptic stimulation or bath application of NMDA receptor agonists. Electrophysiological approach to LTD induction requires specialized equipment, while bath applications limit productivity, as only one neuron per sample may be recorded. Here, we present a simple and effective protocol for pharmacological modeling of LTD in primary cultured neurons. This approach relies on highly localized iontophoretic application of NMDA, which induces LTD in individual cells, enhancing experimental throughput. We have analyzed spatio-temporal patterns of iontophoretic drug delivery and demonstrated how this technique may be combined with electrophysiological and live-cell imaging approaches to investigate LTD-related changes in synaptic strength and Ca2+-dependent signaling of neuronal Ca2+ sensor proteins.

0 Q&A 330 Views Apr 20, 2025

The advent of geroscience engendered the development of approaches to quantify the aging process and estimate biological age on an individual level. Recognizing that declines observed in aging are not only physical but also social led us to develop a mouse Social Frailty Index (mSFI) designed to quantify age-related impairments of social functioning in mice. The mSFI consists of seven behavioral assays that measure essential facets of social behavioral functioning in mice: social communication, social interaction, and social functional ability. The assays that comprise the mSFI are all minimally disruptive, relatively simple to execute, and optimized for compatibility with longitudinal studies utilizing experimental interventions relevant to geroscience. The mSFI is conducted over AM and PM sessions spanning a maximum of 3.5 days, using materials common to most animal facilities. The data for all assays is obtained observationally, manually recorded, and entered into predefined template sheets that automate the computation of the mSFI. We have demonstrated the validity and applicability of the mSFI across multiple laboratory sites and experiments. This index has proven to discriminate between differential trajectories of biological aging driven by sex, progeria, or social stress-relevant contexts. The mSFI represents a novel index to quantify trajectories of biological aging in mice, and its application may help elucidate the social dimensions of the aging process.

0 Q&A 287 Views Apr 20, 2025

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.

0 Q&A 277 Views Mar 20, 2025

The early detection of meningitis pathogens—including Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae, and Klebsiella pneumoniae—through point-of-care (POC) systems is essential for mitigating the risk of neurological damage, enhancing patient outcomes, and facilitating prompt clinical decision-making. Nucleic acid amplification testing (NAAT) is a promising tool for improving the diagnosis process of bacterial pathogens associated with brain inflammation. This is due to its high sensitivity, rapidity, and compatibility with portable diagnostic platforms, making it particularly suitable for POC applications. This protocol introduces an innovative diagnostic approach designed to function effectively without the need for advanced laboratory equipment. By leveraging dual-priming isothermal amplification (DAMP), the assay uses custom internal primers to enhance specificity and minimize false results. Brilliant Green is used in this assay for fluorescence detection due to its availability, high fluorescence level, and optimal sample-to-background (S/B) ratio. The assay demonstrated excellent specificity, absence of false positives, sensitivity comparable to loop-mediated isothermal amplification (LAMP), and a high S/B ratio.

0 Q&A 312 Views Mar 20, 2025

Stroke is a worldwide leading cause of death and long-term disability, with ischemic strokes making up approximately 85% of all cases. There is a significant need for an ideal animal model that accurately replicates the disease’s pathology to study the molecular mechanisms of brain injury. Various experimental models have been created to induce middle cerebral artery occlusion (MCAO), including intraluminal MCAO, photothrombotic models, endothelin-1 injections, and electrocoagulation. However, these often result in large infarct or lesion volumes accompanied by considerable variability. In this study, we present a ministroke model that specifically targets the mouse barrel cortex, making it suitable for investigating the mechanisms of minor strokes and stroke recurrence. In our model, the distal branch of the right middle cerebral artery (MCA), which supplies the sensorimotor cortex, is permanently ligated using 10-0 sutures. This is followed by a 7-min occlusion of the bilateral common carotid arteries (CCAs) and subsequent reperfusion. This approach produces a mild stroke characterized by small and consistent lesion volumes and very low mortality rates. A well-trained experimenter can achieve nearly zero mortality with this technique. Furthermore, this model of localized ischemia induces lesions in the functionally defined barrel cortex, allowing the use of the vibrissae-evoked forelimb placing test to assess functional outcomes.