Figure 1. Schematic representation of a human brain synapse. Presynaptic molecules are labeled in purple and green, while the postsynaptic density (PSD) is illustrated with red and orange molecules.

Chapter 1

Studying neuronal connections

Brain functions are operated through neural connections between dendritic spines and axon terminals, known as synapses. Synapses operate in a sophisticated and cooperative manner in order to maintain brain function and adapt/respond to stimuli.

Challenges in understanding synapses

The small size of synapses, under 200 nm in size, can hinder the study of the molecular mechanisms regulating neurotransmission. Each synapse is composed of over 200 proteins and a synaptic cleft, across which communication between cell terminals is mediated, that can be as small as 20-40nm (Figure 1).

Understanding the crucial mechanisms that determine neuronal connections in health and disease is limited by multiple factors. Firstly, culture cell lines for certain brain cell types can be difficult to obtain and maintain in vitro, making it challenging to find truly predictive neural models. Currently, the most accessible super-resolution imaging studies use primary cortical and hippocampal cell cultures from mice or rats. Yet these models can be considered artificial environments with low complexity brain architectures. An attractive alternative for neuroscientists is the use of neurons differentiated from iPSCs (induced Pluripotent Stem Cells, generated directly from adult cells), which allows research on human cell lines. However, the lack of disease models necessary for iPCS is limiting research using this approach.

Another limitation in neuroimaging is the availability of labeling tools; available antibodies target the most common and best researched proteins while other players remain unexplored, hindering the detailed analysis of neuronal synapse architecture.

Chapter 2

Modeling disease: single-molecule imaging in brain tissues

Brain tissue complexity in neurodegeneration

Our brain is composed of highly complex and densely packed tissues with specialized functions. An adult human brain contains an estimated 86 billion neurons and a similar number of supporting cells, including astrocytes and microglia. On average, each brain cell is connected to around 25,000 other cells, meaning that tissues are integrated by local intertwined cells that project to distant regions. This makes three-dimensional (3D) visualization of brain tissues a challenging job for neuroscientists.

There are several challenges facing research on brain tissues, which commonly include sample sourcing and preparation, specimen labeling, imaging of thick sections, or 3-dimensional data acquisition, often involving time-consuming and laborious protocols. Current efforts are also concentrating on using stem cell models and molecular tools to mimic brain physiology and better explore the origins of neurological disorders.

Current ground-breaking studies are focusing on the discovery and validation of early-disease biomarkers in human fluids, such as cerebrospinal fluid (CSF), which could help diagnose neurodegenerative diseases at early/pre-symptomatic stages, given that high-sensitivity detection of basal levels of biomarkers is achieved (attomolar concentrations in most cases). This would also contribute to the better understanding of disease mechanisms and allow a better patient stratification prior to therapy. Early clinical trials are currently underway to test how small drugs can block or stimulate key molecular interactions involved in neurodegenerative disease progression.

In recent years, extracellular vesicles (EVs) have also been studied as potential carriers of molecules for intercellular communication within tissues and throughout the body, with their unique ability to cross the blood-brain barrier and facilitate communication between peripheral tissues and the central nervous system (CNS). Read our EV guide for more details on single-molecule imaging solutions for studying extracellular vesicles.

Figure 3. Overview scan of a mouse brain tissue section. Image of a 10µm-thick mouse brain tissue. Nuclei were labeled with DAPI and the Nanoimager overview scan feature was used to identify the structure of interest, the hippocampus. Sample prepared by Robert Hinshaw, Ann Romney Center for Neurologic Diseases, Brigham and Women’s Hospital, Boston.

Chapter 3

Neuro-Nanoimaging

The Nanoimager platform enables single-molecule neuroimaging of cultured neuronal cells and brain tissue sections, with a resolution reaching 20 nm. Combining automation features with super-resolution imaging allows the quick and efficient identification of region of interests to then gather localizations from thousands of synaptic proteins. It also facilitates acquisition of data on single molecule distribution, co-localization, or postsynaptic density profiles.

Supporting both fixed and live sample imaging, researchers can develop their own assays to build a complete spatio-temporal portrait of neuronal pathways. Single-molecule sensitivity means potential for improved, accelerated diagnostics and disease therapies. Understanding how drugs alter neurotransmission mechanisms and kinetics with minimal sample requirements can also translate into robust, fast and efficient drug profiling, in a more cost-efficient manner.

Single-molecule imaging has the potential to help establish and characterize new disease models to enable robust testing of novel therapeutics and facilitate drug development. With its compact design, lack of need for a dark room or additional infrastructure, the Nanoimager is the most accessible super-resolution tool ever designed. To learn more about the microscope and ONI, please visit the Nanoimager page.

Summary

Single-molecule imaging opens new possibilities in neuroimaging field. It gives researchers new tools to:

  • Determine synaptic architecture in healthy and disease-affected tissues
  • Follow protein dynamics in living brains by imaging different labels simultaneously
  • Understand the molecular mechanisms of tissue neurodegeneration
  • Identify hallmark disease-linked markers to improve early diagnosis and provide new drug targets
  • Speed up drug development and characterization of neurodegenerative, psychiatric or neurodevelopmental disorders

 

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