神经科学

Amylin is an amyloidogenic neuroendocrine hormone co-synthesized and co-secreted with insulin from the pancreas. It readily crosses the blood–brain barrier and synergistically forms mixed amyloid plaques with β-amyloid (Aβ) in brain parenchyma. Parenchymal amylin-Aβ plaques are found in both sporadic and early-onset familial Alzheimer’s disease (AD), yet their (patho)physiological role remains elusive, particularly due to a lack of detection modalities for these mixed plaques. Previously, we developed an enzyme-linked immunosorbent assay (ELISA) capable of detecting amylin-Aβ hetero-oligomers in brain lysate and blood using a polyclonal anti-amylin antibody to capture hetero-oligomers and a monoclonal anti-Aβ mid-domain detection antibody combination. This combination allows for the recognition of distinct amylin epitopes, which remain accessible after amylin-Aβ oligomerization has begun, and precise detection of Aβ epitopes available after oligomer formation. The utility of this assay is evidenced in our previous report, wherein differences in hetero-oligomer content in brain tissue from patients with and without AD and patients with and without diabetes were distinguished. Additionally, using AD model rats, we provided evidence that our assay can be employed for the detection of amylin-Aβ in blood. This assay and protocol are important innovations in the field of AD research because they meet an unmet need to detect mixed amyloid plaques that, if targeted therapeutically, could reduce AD progression and severity.

The organ of Corti, located in the inner ear, is the primary organ responsible for animal hearing. Each hair cell has a V-shaped or U-shaped hair bundle composed of actin-filled stereocilia and a kinocilium supported by true transport microtubules. Damage to these structures due to noise exposure, drug toxicity, aging, or environmental factors can lead to hearing loss and other disorders. The challenge when examining auditory organs is their location within the bony labyrinth and their small and fragile nature. This protocol describes the dissection procedure for the cochlear organ, followed by confocal imaging of immunostained endogenous and fluorescent proteins. This approach can be used to understand hair cell physiology and the molecular mechanisms required for normal hearing.

Magnetic resonance imaging (MRI) is an invaluable method of choice for anatomical and functional in vivo imaging of the brain. Still, accurate delineation of the brain structures remains a crucial task of MR image evaluation. This study presents a novel analytical algorithm developed in MATLAB for the automatic segmentation of cerebrospinal fluid (CSF) spaces in preclinical non-contrast MR images of the mouse brain. The algorithm employs adaptive thresholding and region growing to accurately and repeatably delineate CSF space regions in 3D constructive interference steady-state (3D-CISS) images acquired using a 9.4 Tesla MR system and a cryogenically cooled transmit/receive resonator. Key steps include computing a bounding box enclosing the brain parenchyma in three dimensions, applying an adaptive intensity threshold, and refining CSF regions independently in sagittal, axial, and coronal planes. In its original application, the algorithm provided objective and repeatable delineation of CSF regions in 3D-CISS images of sub-optimal signal-to-noise ratio, acquired with (33 μm)³ isometric voxel dimensions. It allowed revealing subtle differences in CSF volumes between aquaporin-4-null and wild-type littermate mice, showing robustness and reliability. Despite the increasing use of artificial neural networks in image analysis, this analytical approach provides robustness, especially when the dataset is insufficiently small and limited for training the network. By adjusting parameters, the algorithm is flexible for application in segmenting other types of anatomical structures or other types of 3D images. This automated method significantly reduces the time and effort compared to manual segmentation and offers higher repeatability, making it a valuable tool for preclinical and potentially clinical MRI applications.

Drosophila larvae exhibit rolling motor behavior as an escape response to avoid predators and painful stimuli. We introduce an accessible method for applying optogenetics to study the motor circuits driving rolling behavior. For this, we simultaneously implement the Gal4-UAS and LexA-Aop binary systems to express two distinct optogenetic channels, GtACR and Chrimson, in motor neuron (MN) subsets and rolling command neurons (Goro), respectively. Upon exposure to white LED light, Chrimson permits the influx of positive ions into Goro neurons, leading to depolarization, whereas GtACR mediates chloride influx into MNs, resulting in hyperpolarization. This method allows researchers to selectively activate certain neurons while simultaneously inhibiting others within a circuit of interest, offering a unique advantage over current optogenetic approaches, which often utilize a single type of optogenetic actuator. Here, we provide a detailed protocol for the dual silencing-activation approach using GtACR and Chrimson optogenetic channels and present a robust methodological framework for investigating the neuromuscular basis of rolling in larvae. Our cost-effective and scalable approach utilizes readily accessible equipment and can be applied to study other locomotor behaviors in Drosophila larvae, thereby enhancing our understanding of the neural circuit mechanisms underlying sensorimotor transformation.

Neuroscience incorporates manipulating neuronal circuitry to enhance the understanding of intricate brain functions. An effective strategy to attain this objective entails utilizing viral vectors to induce varied gene expression by delivering transgenes into brain cells. Here, we combine the use of transgenic mice, neonatal transduction with adeno-associated viral constructs harboring inhibitory designer receptor exclusively activated by designer drug (DREADD) gene, and the DREADD agonist clozapine N-oxide (CNO). In this way, a chemogenetic approach is employed to suppress neuronal activity in the region of interest during a critical developmental window, with subsequent investigation into its effects on the neuronal circuitry in adulthood.

The neuromuscular junction (NMJ) is an interface between motor neurons and skeletal muscle fibers, facilitating the transmission of signals that initiate muscle contraction. Its pivotal role lies in ensuring efficient communication between the nervous system and muscles, allowing for precise and coordinated movements essential for everyday activities and overall motor function. To provide insights into neuromuscular disease and development, understanding the physiology of NMJ is essential. We target acetylcholine receptors (AChR) by immunofluorescence assay (IFA) with α-bungarotoxin (BTX; snake venom neurotoxins binding to AChR) to visualize and quantify the NMJ. Changes in AChR distribution or structure can indicate alterations in receptor density, which may be associated with neuromuscular disorders or conditions that affect synaptic transmission. This protocol provides the methodology for isolating and longitudinally sectioning gastrocnemius muscle for AChR-targeted IFA for confocal microscopy and performing quantitative analysis of NMJs.

In vivo brain imaging, using a combination of genetically encoded Ca²⁺ indicators and gradient refractive index (GRIN) lens, is a transformative technology that has become an increasingly potent research tool over the last decade. It allows direct visualisation of the dynamic cellular activity of deep brain neurons and glia in conscious animals and avoids the effect of anaesthesia on the network. This technique provides a step change in brain imaging where fibre photometry combines the whole ensemble of cellular activity, and multiphoton microscopy is limited to imaging superficial brain structures either under anaesthesia or in head-restrained conditions. We have refined the intravital imaging technique to image deep brain nuclei in the ventral medulla oblongata, one of the most difficult brain structures to image due to the movement of brainstem structures outside the cranial cavity during free behaviour (head and neck movement), whose targeting requires GRIN lens insertion through the cerebellum—a key structure for balance and movement. Our protocol refines the implantation method of GRIN lenses, giving the best possible approach to image deep extracranial brainstem structures in awake rodents with improved cell rejection/acceptance criteria during analysis. We have recently reported this method for imaging the activity of retrotrapezoid nucleus and raphe neurons to outline their chemosensitive characteristics. This revised method paves the way to image challenging brainstem structures to investigate their role in complex behaviours such as breathing, circulation, sleep, digestion, and swallowing, and could be extended to image and study the role of cerebellum in balance, movement, motor learning, and beyond.

Key features

• We developed a protocol that allows imaging from brainstem neurons and glia in freely behaving rodents.

• Our refined method of GRIN lenses implantation and cell sorting approach gives the highest number of cells with the least postoperative complications.

• The revised deep brainstem imaging method paves way to understand complex behaviours such as cardiorespiratory regulation, sleep, swallowing, and digestion.

• Our protocol can be implemented to image cerebellar structures to understand their role in key functions such as balance, movement, motor learning, and more.

Graphical overview

The blood–brain barrier (BBB) is a major obstacle to the diagnostics and treatment of many central nervous system (CNS) diseases. A prime example of this challenge is seen in glioblastoma (GBM), the most aggressive and malignant primary brain tumor. The BBB in brain tumors, or the blood–brain–tumor barrier (BBTB), prevents the efficient delivery of most therapeutics to brain tumors. Current strategies to overcome the BBB for therapeutic delivery, such as using hyperosmotic agents (mannitol), have impeded progress in clinical translation limited by the lack of spatial resolution, high incidences of complications, and potential for toxicity. Focused ultrasound combined with intravenously administered microbubbles enables the transient disruption of the BBB and has progressed to early-phase clinical trials. However, the poor survival with currently approved treatments for GBM highlights the compelling need to develop and validate treatment strategies as well as the screening for more potent anticancer drugs. In this protocol, we introduce an optical method to open the BBTB (OptoBBTB) for therapeutic delivery via ultrashort pulse laser stimulation of vascular targeting plasmonic gold nanoparticles (AuNPs). Specifically, the protocol includes the synthesis and characterization of vascular-targeting AuNPs and a detailed procedure of optoBBTB. We also report the downstream characterization of the drug delivery and tumor treatment efficacy after BBB modulation. Compared with other barrier modulation methods, our optical approach has advantages in high spatial resolution and minimally invasive access to tissues. Overall, optoBBTB allows for the delivery of a variety of therapeutics into the brain and will accelerate drug delivery and screening for CNS disease treatment.

Key features

• Pulsed laser excitation of vascular-targeting gold nanoparticles non-invasively and reversibly modulates the blood–brain barrier permeability.

• OptoBBTB enhances drug delivery in clinically relevant glioblastoma models.

• OptoBBTB has the potential for drug screening and evaluation for superficial brain tumor treatment.

Graphical overview

The inferior colliculus (IC) is an important processing center in the auditory system, which also receives non-auditory sensory input. The IC consists of several subnuclei whose functional role in (non-) auditory processing and plastic response properties are best approached by studying awake animals, preferably in a longitudinal fashion. The increasing use of mice in auditory research, the availability of genetic models, and the superficial location of the IC in the mouse have made it an attractive species for studying IC function. Here, we describe a protocol for exposing the mouse IC for up to a few weeks for in vivo imaging or electrophysiology in a stable manner. This method allows for a broader sampling of the IC while maintaining the brain surface in good quality and without reopening the craniotomy. Moreover, as it is adaptable for both electrophysiological recordings of the entire IC and imaging of the dorsal IC surface, it can be applied to answer a multitude of questions.

Key features

• A surgical protocol for long-term physiological recordings from the same or separate neuronal populations in the inferior colliculus.

• Optimized for awake in vivo experiments in the house mouse (Mus musculus).

Recent advancements in chemogenetic tools, such as designer receptors exclusively activated by designer drugs (DREADDs), allow the simultaneous manipulation of activity over a specific, broad brain region in nonhuman primates. However, the introduction of DREADDs into large and complexly shaped cortical sulcus regions of macaque monkeys is technically demanding; previously reported methods are time consuming or do not allow the spatial range of expression to be controlled. In the present report, we describe the procedure for an adeno-associated viral vector (AAV2.1) delivery via handheld injections into the dorsolateral prefrontal cortex (Brodmann’s area 9/46) of macaque monkeys, with reference to pre-scanned anatomical magnetic resonance images. This procedure allows the precise delivery of DREADDs to a specific cortical region.

Key features

• This article describes the procedures for injecting viral vectors encoding functional proteins for chemogenetic manipulation into targeted cortical sulcus regions.

• The protocol requires magnetic resonance imaging for the accurate estimation of the injection sites prior to surgery.

• Viral vector solutions are injected using a handheld syringe under microscopic guidance.

• This protocol allows for the precise introduction of designer receptors exclusively activated by designer drugs (DREADDs) to large and complex cortical regions.