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Dec 1, 2024

Snapshots of a Synapse

In episode 78 of Neuroverse, Dr Rachel Jackson, a research associate at the Centre for Developmental Neurobiology in Kings College London, discusses the molecular complexities of synapses: those crucial points of communication between neurons. Her work aims to untangle what goes wrong at synapses in neurological diseases. To do so, she visualises synaptic proteins and neurotransmitter release in real time, using state-of-the-art imaging methods, which gives a view into these tiny yet very important structures.


Synapses are the connections between neurons, where electrical signals are converted to chemical signals and back to electrical

 

Synapses are crucial to every process in the brain. They are specialized junctions between neurons where information is transmitted from one neuron to another by chemical and electrical signals. In this way, synapses allow everything from sensory perception to memory formation and cognitive processing. At a typical synapse, neurotransmitters are contained within vesicles of the presynaptic neuron. Just before their release, as the action potential or electrical signal reaches, there is an influx of calcium into the neuron. This causes vesicles to fuse with the membrane and release their neurotransmitters through the synaptic cleft; these chemicals bind to specific receptors on the postsynaptic neuron and transform back into an electrical signal.


 

Calcium Influx: A Deeper Look into Synaptic Processes


Since synapses were first discovered as the unit of communication in the brain, a lot has been discovered about their structure and types, such as glutamate being the major excitatory neurotransmitter and GABA being the major inhibitory neurotransmitter. However, a comprehensive understanding of their function remains to be obtained. Central to this lies an intricate process: neurotransmitter release, which is a complex play-off of several factors, including calcium ion flux. This influx plays a crucial role of turning chemical signals into electrical signals: the presence of voltage-sensitive calcium ion channels embedded in the membrane enables electrical signals to induce an influx of calcium.


The process of exocytosis involves the physical movement of vesicles containing neurotransmitters. Calcium enables vesicles to fuse to the membrane and release neurotransmitter across the synaptic cleft to receptors on the postsynaptic side.

 

This influx of calcium is critical for the fusion of vesicles to the membrane to allow for neurotransmitter release, termed vesicular exocytosis. In addition to this, researchers have shown that the size of the vesicular pool that is involved in exocytosis, known as the readily releasable pool (RRP), and how likely it is that vesicles will be released depends on the amount of calcium at the presynaptic terminal. Changing the composition of calcium channels at the presynaptic terminal has also been shown to affect the release of neurotransmitters, proving that not only the number but also the spatial organisation of calcium channels is absolutely important for proper synaptic functioning. This spatial organisation, together with the positioning of docked synaptic vesicles, synergistically influence the probability and dynamics of synaptic release. There is also a huge variety of subtypes of voltage-gated calcium channels present at synapses, each with a different physiological profile (how quickly they open or inactivate), further adding to the complexity of synaptic transmission.

 

Tools to Visualize Synaptic Connections


Understanding the detailed architecture and dynamics of synapses is an important aspect of deciphering how neurons communicate. One of the most innovative techniques used for this purpose is 3D dSTORM (direct Stochastic Optical Reconstruction Microscopy), a form of super-resolution microscopy that relies on the labelling of specific synaptic proteins with fluorophores. This technique can allow the visualisation of proteins located at specialised regions where neurotransmitter release occurs- the presynaptic active zone. Scientists can also closely map the localization and density of synaptic proteins at the pre- and postsynaptic terminals, which has revealed the existence of trans-synaptic nanocolumns- vertical arrangements of proteins that enhance the clustering of synaptic machinery. Techniques such as dSTORM are important for understanding how the spatial arrangement of proteins influences synaptic efficacy and plasticity. Misalignment of pre- and postsynaptic proteins can occur following the acute manipulation of neuronal activity experimentally. This manipulation may become a useful model for dissecting mechanisms underlying synaptic plasticity in response to changes in activity.


Super-resolution imaging (one method being dSTORM) enables synaptic proteins to be imaged at a much better resolution compared to conventional microscopy methods

 

In addition to imaging techniques, biochemical approaches can further elucidate the composition and functional characteristics of synaptic structures. One such example is the isolation of synaptosomes, which are isolated nerve terminals that retain many properties of intact synapses. Biochemical assays can be used on synaptosomes to read out RNA or protein levels, protein-protein interactions, and other properties of biochemical pathways such as receptor signalling. This is useful for quantifying specific proteins of interest and determining the ways in which synaptic activity, pharmacological intervention, or cellular disease states affect synaptic function.


Synaptosomes are isolated nerve terminals with an intact pre- and postsynaptic compartment that can be studied using biochemical techniques

Image from (Evans, 2015; doi: 10.1101/pdb.top074450)


Synapses Out of Form: Disorders Arising From Faulty Synapses

 

Structural and functional synaptic misalignment has been linked to several neurodevelopmental and neurodegenerative diseases, including autism, schizophrenia, amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). These are most commonly associated with genetic mutations that affect synaptic proteins, disrupting the intricate organization of synaptic components at the nanoscale. These mutations can be particularly classified into three major types; namely, those that influence transcriptional regulation (which is key for the production of RNA and protein), synaptic signalling, and synaptic scaffolding (maintaining synaptic structure). This has potential to cause a cascade effect on synapse development and maintenance.


Synapses are rich in proteins that together create a structural scaffold


One example of an important synaptic organising protein is UNC13A, which acts by facilitating vesicle fusion to the presynaptic membrane during the process of neurotransmitter release. Disruption of UNC13A leads to deficits in synaptic efficacy and has been linked to the degeneration of motor neurons in ALS/FTD. Imaging techniques have shown that UNC13A is optimally positioned to regulate neurotransmitter release: it is located close to voltage-gated calcium channels, and interacts with many other scaffolding proteins. Loss of function of UNC13A, in diseased neurons from ALS/FTD patients, has not only been shown to influence presynaptic mechanisms, but also alters the clustering of receptors on the postsynaptic cell. Ultimately, motor neurons become susceptible to further degeneration due to the accumulation of disruptions.

 

Synaptic Connections: From Lab to Life

 

In recent years, therapies that target synaptic mechanisms have emerged as a potentially effective intervention against neurodegenerative diseases such as ALS and FTD. Unlike traditional pharmacological interventions that target clinical symptoms, antisense oligonucleotides (ASOs) make up a relatively novel way to target synaptic deficits. ASOs are short synthetic strands of nucleotides (what DNA and RNA is made of) that can bind to RNA to influence protein synthesis. Binding of ASOs to RNA can alter gene expression directly by promoting the degradation of faulty RNA, or change how RNA is processed by acting on splicing to either increase or decrease expression. ASOs that influence the production of UNC13A protein, for example, could offer a way to restore synaptic function in ALS/FTD. Thus, gaining insight into synaptic function can offer new solutions to target neurological disorders from their source.

Antisense oligonucleotides (ASOs) bind to mRNA and can increase or decrease protein synthesis

 

Conclusions


Overall, it is clear that nanoscale dynamics at the synapse is crucial for the proper functioning of neurons, neural networks, and cognition. Synaptic research is advancing rapidly, driven by innovative techniques like genetically encoded reporters, super-resolution microscopy, and live imaging in real time. This array of tools provides an unprecedented view into both the functionality and structures of synapses. The continued decoding of synaptic structure and function could open new therapeutic avenues aimed at preserving—and potentially restoring—synaptic functions, promising to change the landscape of treatment for neurological diseases.


Listen to the episode here to find out more!


This article was written by Purnima BR and edited by Clara Lenherr

Purnima is Master's student in Cognitive Neuroscience at the University of Sheffield who is enthusiastic about the neural basis of cognition, and how advances in diagnostic visualization can help inform treatments for neuropsychiatric conditions.

 

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