神经科学

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.

Brain organoids have been widely used to study diseases and the development of the nervous system. Many reports have investigated the application of brain organoids, but most of these models lack vascular structures, which play essential roles in brain development and neurological diseases. The brain and blood vessels originate from two different germ layers, making it difficult to induce vascularized brain organoids in vitro. We developed this protocol to generate brain-specific blood vessel and cerebral organoids and then fused them at a specific developmental time point. The fused cerebral organoids exhibited robust vascular network-like structures, which allows simulating the in vivo developmental processes of the brain for further applications in various neurological diseases.

Key Features

• Culturing vascularized brain organoids using human embryonic stem cells (hESCs).

• The new approach generates not only neural cells and vessel-like networks but also brain-resident microglia immune cells in a single organoid.

Graphical overview

An emerging body of behavioural studies indicates that regular swimming in cold water has positive effects on mental health and wellbeing, such as reducing fatigue, improving mood, and lessening depressive symptoms. Moreover, some studies reported immediate effects of cold-water immersion (CWI) on elevating mood and increasing a positive emotional state. However, the neural mechanisms underlying these effects remain largely unknown. The lack of studies using neuroimaging techniques to investigate how a whole-body CWI affects neural processes has partly resulted from the lack of a tested experimental protocol. Previous protocols administered tonic limb cooling (1–10 °C) while recording functional magnetic resonance (fMRI) signals. However, using very low water temperature constitutes points of contrast to painful experiences that are different from what we experience after a whole-body head-out CWI. In our protocol, healthy adults unhabituated to cold water were scanned twice: immediately before (pre-CWI) and after (post-CWI) immersion in cold water (water temperature 20 °C) for 5 min. We recorded cardiac and ventilatory responses to CWI and assessed self-reported changes in positive and negative affects. Our protocol showed reliable changes in brain connectivity after a short exposure to cold water, thus enabling its use as a tested experimental framework in future studies.

Graphical overview

The zebrafish retina is a canonical vertebrate retina. Since the past few years, with the continually growing genetic toolbox and imaging techniques, zebrafish plays a crucial role in retinal research. This protocol describes a method to quantitatively evaluate the expression of Arrestin3a (Arr3a) and G-protein receptor kinase7a (Grk7a) in the adult zebrafish retina at protein levels by infrared fluorescence western blot. Our protocol can be easily adapted to measure protein levels in additional zebrafish tissues.

Molecular characterization of different cell types in rodent brains is a widely used and important approach in neuroscience. Fluorescent detection of transcripts using RNAscope (ACDBio) has quickly became a standard in situ hybridization (ISH) approach. Its sensitivity and specificity allow for the simultaneous detection of between three and forty-eight low abundance mRNAs in single cells (i.e., multiplexing or hiplexing), and, in contrast to other ISH techniques, it is performed in a shorter amount of time. Manual quantification of transcripts is a laborious and time-consuming task even for small portions of a larger tissue section. Herein, we present a protocol for creating high-quality images for quantification of RNAscope-labeled neurons in the rat brain. This protocol uses custom-made scripts within the open-source software QuPath to create an automated workflow for the careful optimization and validation of cell detection parameters. Moreover, we describe a method to derive mRNA signal thresholds using negative controls. This protocol and automated workflow may help scientists to reliably and reproducibly prepare and analyze rodent brain tissue for cell type characterization using RNAscope.

Graphical abstract

Targeting receptor-mediated transcytosis (RMT) is a successful strategy for drug delivery of biologic agents across the blood-brain barrier (BBB). The recent development of human BBB organoid models is a major advancement to help characterize the mechanisms of RMT and thus accelerate the design of brain delivery technologies. BBB organoids exhibit self-organization, which resembles the architecture of the neurovascular unit, and low paracellular permeability, due to the formation of tight junctions between endothelial cells. However, current methods of organoid generation have low throughput, exhibit substantial heterogeneity across experiments, and require extensive manual handling. These limitations prevent the use of BBB organoids as a screening tool for discovery and optimization of therapeutic molecules. In this protocol, we use hydrogel-based arrays to generate human BBB organoids, with a 35-fold increase in organoid yield as compared to previous protocols using 96-well plates. We incubate BBB organoid arrays with monoclonal antibody-based constructs and use a custom semi-automated imaging assay to assess RMT within the organoid core. The experimental and analytical tools described in this protocol provide a scalable platform that can be incorporated in the early stages of drug discovery to accelerate the development and optimization of brain delivery technologies to cross the BBB.

Thermotaxis behaviors in C. elegans exhibit experience-dependent plasticity of thermal preference memory. This behavior can be assayed either at population level, on linear temperature gradients, or at the individual animal level, by radial isothermal or microfluidic tracking of orientation. These behaviors are low-throughput as well as variable, due to the inherent sensitivity to environmental perturbations. To facilitate reproducible studies, we describe an updated apparatus design that enables simultaneous runs of three thermal preference assays, instead of single-run assays described previously. By enabling parallel runs of control and experimental conditions, this set-up enables more throughput and rigorous assessment of behavioral variability.

Optogenetics has the potential to transform the study of the peripheral nervous system (PNS), but the complex anatomy of the PNS poses unique challenges for the focused delivery of light to specific tissues. This protocol describes the fabrication of a wireless telemetry system for studying peripheral sensory pathways. Unlike existing wireless approaches, the low-power wireless telemetry offers organ specificity via a sandwiched pre-curved tether, and enables high-throughput analysis of behavioral experiments with a channel isolation strategy. We describe the technical procedures for the construction of these devices, the wireless power transmission (TX) system with antenna coils, and their implementation for in vivo experimental applications. In total, the timeline of the procedure, including device fabrication, implantation, and preparation to begin in vivo experimentation can be completed in ~2-4 weeks. Implementation of these devices allows for chronic (>1 month) wireless optogenetic manipulation of peripheral neural pathways in freely behaving animals navigating homecage environments (up to 8).