Pre- and Postembedding Immunoelectron Microscopy to Analyze the Central Nervous System.

Immunocytochemistry, a method of delineating the subcellular localization of target proteins, was developed from immunohistochemistry. In principle, proteins are labeled using an antigen-antibody reaction. In order to observe under an electron microscope, the reaction product must scatter the electron beam with sufficient contrast while it is necessary to have an amplifying label that can withstand the observation. We have some detailed tips on making electron microscope samples to achieve this objective, and we would be happy to help you.

Globus pallidus is not independent from striatal direct pathway neurons: an up-to-date review.

Striatal projection neurons, which are classified into two groups-direct and indirect pathway neurons, play a pivotal role in our understanding of the brain's functionality. Conventional models propose that these two pathways operate independently and have contrasting functions, akin to an "accelerator" and "brake" in a vehicle. This analogy further elucidates how the depletion of dopamine neurons in Parkinson's disease can result in bradykinesia. However, the question arises: are these direct and indirect pathways truly autonomous? Despite being distinct types of neurons, their interdependence cannot be overlooked. Single-neuron tracing studies employing membrane-targeting signals have shown that the majority of direct pathway neurons terminate not only in the output nuclei, but also in the external segment of the globus pallidus (GP in rodents), a relay nucleus of the indirect pathway. Recent studies have unveiled the existence of arkypallidal neurons, which project solely to the striatum, in addition to prototypic neurons. This raises the question of which type of GP neurons receive these striatal axon collaterals. Our morphological and electrophysiological experiments showed that the striatal direct pathway neurons may affect prototypic neurons via the action of substance P on neurokinin-1 receptors. Conversely, another research group has reported that direct pathway neurons inhibit arkypallidal neurons via GABA. Regardless of the neurotransmitter involved, it can be concluded that the GP is not entirely independent of direct pathway neurons. This review article underscores the intricate interplay between different neuronal pathways and challenges the traditional understanding of their independence.

Volume electron microscopy for genetically and molecularly defined neural circuits.

The brain networks responsible for adaptive behavioral changes are based on the physical connections between neurons. Light and electron microscopy have long been used to study neural projections and the physical connections between neurons. Volume electron microscopy has recently expanded its scale of analysis due to methodological advances, resulting in complete wiring maps of neurites in a large volume of brain tissues and even entire nervous systems in a growing number of species. However, structural approaches frequently suffer from inherent limitations in which elements in images are identified solely by morphological criteria. Recently, an increasing number of tools and technologies have been developed to characterize cells and cellular components in the context of molecules and gene expression. These advancements include newly developed probes for visualization in electron microscopic images as well as correlative integration methods for the same elements across multiple microscopic modalities. Such approaches advance our understanding of interactions between specific neurons and circuits and may help to elucidate novel aspects of the basal ganglia network involving dopamine neurons. These advancements are expected to reveal mechanisms for processing adaptive changes in specific neural circuits that modulate brain functions.

Anterograde trans-neuronal labeling of striatal interneurons in relation to dopamine neurons in the substantia nigra pars compacta.

Recent advances in neural tracing have unveiled numerous neural circuits characterized by brain region and cell type specificity, illuminating the underpinnings of specific functions and behaviors. Dopaminergic (DA) neurons in the midbrain are highly heterogeneous in terms of gene and protein expression and axonal projections. Different cell types within the substantia nigra pars compacta (SNc) tend to project to the striatum in a cell-type-dependent manner characterized by specific topography. Given the wide and dense distribution of DA axons, coupled with a combination of synaptic and volume transmission, it remains unclear how DA release is spatially and temporally regulated, to appropriately achieve specific behaviors and functions. Our hypothesis posits that hidden rules governing synapse formation between pre-synaptic DA neuron types and striatal neuron types may modulate the effect of DA at a single-cell level. To address this conjecture, we employed adeno-associated virus serotype 1 (AAV1) to visualize the neural circuitry of DA neurons. AAV1 has emerged as a potent anatomical instrument capable of labeling and visualizing pre- and post-synaptic neurons simultaneously through anterograde trans-synaptic labeling. First, AAV1-Cre was injected into the SNc, resulting in Cre expression in both medium spiny neurons and interneurons in the striatum. Due to the potential occurrence of the retrograde transfer of AAV1, only striatal interneurons were considered for trans-synaptic or trans-neuronal labeling. Interneuron types expressing parvalbumin, choline acetyltransferase, somatostatin, or nitrogen oxide synthase exhibited Cre expression. Using a combination of AAV1-Cre and Cre-driven fluorophore expressing AAVs, striatal interneurons and the axons originating from the SNc were visualized in distinct colors. Using immunofluorescence against neurotransmitter transporters, almost all axons in the striatum visualized using this approach were confirmed to be dopaminergic. Moreover, individual DA axons established multiple appositions on the somata and proximal dendrites of interneurons. This finding suggests that irrespective of the extensive and widespread axonal arborization of DA neurons, a particular DA neuron may exert a significant influence on specific interneurons. Thus, AAV1-based labeling of the DA system can be a valuable tool to uncover the concealed rules governing these intricate relationships.

descSPIM: an affordable and easy-to-build light-sheet microscope optimized for tissue clearing techniques.

Despite widespread adoption of tissue clearing techniques in recent years, poor access to suitable light-sheet fluorescence microscopes remains a major obstacle for biomedical end-users. Here, we present descSPIM (desktop-equipped SPIM for cleared specimens), a low-cost ($20,000-50,000), low-expertise (one-day installation by a non-expert), yet practical do-it-yourself light-sheet microscope as a solution for this bottleneck. Even the most fundamental configuration of descSPIM enables multi-color imaging of whole mouse brains and a cancer cell line-derived xenograft tumor mass for the visualization of neurocircuitry, assessment of drug distribution, and pathological examination by false-colored hematoxylin and eosin staining in a three-dimensional manner. Academically open-sourced ( https://github.com/dbsb-juntendo/descSPIM ), descSPIM allows routine three-dimensional imaging of cleared samples in minutes. Thus, the dissemination of descSPIM will accelerate biomedical discoveries driven by tissue clearing technologies.