Neuroscience

An improved correlative light and electron microscopy (CLEM) method has recently been introduced and successfully employed to identify and analyze protein inclusions in cultured cells as well as pathological proteinaceous deposits in postmortem human brain tissues from individuals with diverse neurodegenerative diseases. This method significantly enhances antigen preservation and target registration by replacing conventional dehydration and embedding reagents. It achieves an optimal balance of sensitivity, accuracy, efficiency, and cost-effectiveness compared to other current CLEM approaches. However, due to space constraints, only a brief overview of this method was provided in the initial publication. To ensure reproducibility and facilitate widespread adoption, the author now presents a detailed, step-by-step protocol of this optimized CLEM technique. By enhancing usability and accessibility, this protocol aims to promote broader application of CLEM in neurodegenerative disease research.

The global burden of stroke has increased in the past several decades, and post-stroke epilepsy (PSE) is a common complication. Contrasted with the advancement in knowledge of stroke pathophysiology, the exact pathogenesis of PSE is unclear. Various animal stroke models have been utilized to investigate the underlying mechanisms of PSE, but the success rate of PSE induction is low. To address this limitation, a novel PSE model was established in the rat by inducing status epilepticus using lithium-pilocarpine one week after photothrombotic stroke. Successful indication of status epilepticus and mortality rate at three days after status epilepticus were the main measurements. Potential usefulness of this model was also illustrated by preliminary results on locomotor activity, exploratory behavior, and anxiety level evaluated using the open-field test, as well as mossy fiber sprouting (MFS) in the hippocampal dentate granule cells using Zinc transporter 3 immunofluorescence staining at 8 weeks after PSE induction. This novel composite method of PSE induction may facilitate future studies on the pathogenesis and treatment of PSE.

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.

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.

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.

The neuromuscular junction (NMJ) is a peripheral synaptic connection between a lower motor neuron and skeletal muscle fibre that enables muscle contraction in response to neuronal stimulation. NMJ dysfunction and morphological abnormalities are commonly observed in neurological conditions, including amyotrophic lateral sclerosis, Charcot–Marie–Tooth disease, and spinal muscular atrophy. Employing precise and reproducible techniques to visualise NMJs in mouse models of neuromuscular disorders is crucial for uncovering aspects of neuropathology, revealing disease mechanisms, and evaluating therapeutic approaches. Here, we present a method for dissecting the deep lumbrical and flexor digitorum brevis (FDB) muscles of the mouse hind paw and describe the process of whole-mount immunofluorescent staining for morphological analysis of NMJs. Similar whole-mount techniques have been applied to other muscles, such as the diaphragm; however, dense connective tissue in adult samples often impedes antibody penetration. Moreover, large hind limb muscles, including the gastrocnemius and tibialis anterior, are commonly used to examine NMJs but require embedding and cryosectioning. These additional steps increase the complexity and duration of the protocol and can introduce sectioning artefacts, including transection of NMJs and disruption of morphology. Using small hind paw muscles enables whole-mounting, which completely eliminates the requirement for embedding and cryosectioning. As a result, the entire neuromuscular innervation pattern can be visualised, allowing a more accurate assessment of NMJ development, denervation, and regeneration in mouse models of neurological disease and nerve injury, which can be applied across all postnatal ages.

Reconstructing single-neuron projectomes is essential for mapping the mesoscopic connectome and eventually for understanding brain-wide connectivity and diverse brain functions. The combination of sparse labeling techniques and large-scale and high-resolution optical imaging technologies has been revolutionizing the brain-wide reconstruction of single-neuron morphologies, as exemplified by the dataset for over 10,100 single-neuron projectomes of hippocampal neurons. Here, we illustrate a comprehensive protocol for large-scale single-neuron reconstruction in the mouse brain. This includes key steps and examples in imaging data preprocessing, neurite tracing, and registration into a template brain. These procedures enable efficient and accurate large-scale morphological reconstruction of single neurons in the mouse brain.

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.

Changes in neuronal conduction are common in disease states affecting peripheral nerves. These alterations can significantly impact nerve function and lead to sensorimotor disabilities. In vivo electromyography recording is a well-established electrophysiological method that has been used for decades to assess sensory and motor functions in the nervous system. Nerve studies are challenging to conduct in vivo in rodents, and the involvement of muscle activity makes it difficult to isolate and assess nerve function independently. This protocol provides a comprehensive guide for accurate ex vivo sciatic nerve dissection and handling from mice. It includes the creation of a three-compartment chamber and the establishment of electrophysiological protocols, which enable differential recordings and the analysis of compound action potentials from various nerve fibers. This setup allows researchers to study the specific effects of drugs and pathologies on nerves from a mechanistic perspective. The setup is a stand-alone apparatus that does not require the use of suction electrodes and the maintenance of negative pressure, which can affect the signal-to-noise ratio and recording stability.

Fluorescence lifetime imaging microscopy (FLIM) is a highly valuable technique in the fluorescence microscopy toolbox because it is essentially independent of indicator concentrations. Conventional fluorescence microscopy analyzes changes in emission intensity. In contrast, FLIM assesses the fluorescence lifetime, which is defined as the time a fluorophore remains in an excited state before emitting a photon. This principle is advantageous in experiments where fluorophore concentrations are expected to change, e.g., due to changes in cell volume. FLIM, however, requires collecting a substantial number of photons to accurately fit distribution plots, which constrains its ability for dynamic imaging. This limitation has recently been overcome by rapidFLIM, which utilizes ultra-low dead-time photodetectors in conjunction with sophisticated rapid electronics. The resulting reduction in dead-time to the picosecond range greatly enhances the potential for achieving high spatio-temporal resolution. Here, we demonstrate the use of multi-photon-based rapidFLIM with the sodium indicator ION NaTRIUM Green-2 (ING-2) for the quantitative, dynamic determination of Na⁺ concentrations in neurons in acute rodent brain tissue slices. We describe the loading of the dye into neurons and present a procedure for its calibration in situ. We show that rapidFLIM not only allows the unbiased determination of baseline Na⁺ concentrations but also allows dynamic imaging of changes in intracellular Na⁺, e.g., induced by inhibition of cellular ATP production. Overall, rapidFLIM, with its greatly improved signal-to-noise ratio and higher spatio-temporal resolution, will also facilitate dynamic measurements using other FLIM probes, particularly those with a low quantum yield.