神经科学

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.

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.

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.

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.

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.

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.

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.

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.

The growth cone is a highly motile tip structure that guides axonal elongation and directionality in differentiating neurons. Migrating immature neurons also exhibit a growth cone–like structure (GCLS) at the tip of the leading process. However, it remains unknown whether the GCLS in migrating immature neurons shares the morphological and molecular features of axonal growth cones and can thus be considered equivalent to them. Here, we describe a detailed method for time-lapse imaging and optical manipulation of growth cones using a super-resolution laser-scanning microscope. To observe growth cones in elongating axons and migrating neurons, embryonic cortical neurons and neonatal ventricular–subventricular zone (V-SVZ)-derived neurons, respectively, were transfected with plasmids encoding fluorescent protein–conjugated cytoskeletal probes and three-dimensionally cultured in Matrigel, which mimics the in vivo background. At 2–5 days in vitro, the morphology and dynamics of these growth cones and their associated cytoskeletal molecules were assessed by time-lapse super-resolution imaging. The use of photoswitchable cytoskeletal inhibitors, which can be reversibly and precisely controlled by laser illumination at two different wavelengths, revealed the spatiotemporal regulatory machinery and functional significance of growth cones in neuronal migration. Furthermore, machine learning–based methods enabled us to automatically segment growth cone morphology from elongating axons and the leading process. This protocol provides a cutting-edge methodology for studying the growth cone in developmental and regenerative neuroscience, being adaptable for various cell biology and imaging applications.

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.