Development of Optical Imaging Tools for Synapse Typing
Restructure of synapse types and alternations of dynamics of neurotransmitters and neuromodulators have been implicated in a number of human neurological and psychiatric diseases, including autism, Alzheimer’s disease, Parkinson’s disease, schizophrenia, and addiction1,2. However, little is known about how molecular compositions define different types and functions of synapses and how different synapses organize in micro- and macroscale to give rise to complex brain functions and disorders, due to lack of appropriate tools to characterize synaptic biomolecules in situ in large scale. Here, I propose a novel research program to develop transformative tools for large-scale mapping of synaptic biomolecules, functional imaging of neurotransmission and neuronal signaling. Specifically, it will evolve along three main themes: 1. Expansion pathology for highly multiplexed, in situ and nanoscale biomolecular imaging. 2. Optogenetic neurotransmitter indicator; 3. in situ imaging tools for endogenous protein-protein interaction; This program will expand on the themes of my previous work on engineering optogenetic indicators for neuronal activities, such as calcium ions3,4 and voltage5,6 indicators, as well as my current work on devising a clinically optimized form of expansion microscopy7,8, which contribute new capabilities to the broader communities of neuroscience, cell biology, pathology and medicine.
Theme 1: Expansion
Anything we do as humans such as thinking, feeling, and hearing would not happen without the neuron. The neuron sends messages throughout the body when the body is in trouble or pain. The neuron is a part of the nervous system which makes the body move. There are only three structures to the neuron; cell body, dendrite, and the axon. With these structures come functions to help the body think, feel, and hear. When these functions don’t work they can cause diseases in which the body needs to be healthy to work. These are the reasons the neuron is important.
Voltage-gated ion channels maintain the concentrations of different ions inside and outside of the neuron cell.
The end of the axon spread into some shorter fibers that have swellings on the ends called synaptic knobs. The synaptic knob has a number of little saclike structures in it called synaptic vesicles. Inside the synaptic vesicles are chemicals hung in fluid, which are molecules of substances called neurotransmitters which are inside a neuron and are going to transmit a message. Neurotransmitter are released into the synapse from synaptic vesicles. The neurotransmitter molecules bind to receptor sites on the releasing neuron and the second neuron or glands or even muscles causing a reaction.
Neurological disorders such as Alzheimer’s disease, autism spectrum disorders (ASD), and schizophrenia impact behavior and cognitive processes. While the symptoms for these diseases vary, they share disruptions in synapse development and spine formation. In the mammalian nervous system, dendritic spines are small, dynamic protrusions on neurons that develop in response to increased presynaptic activity. Although spine dynamics throughout development have been well studied, very little is known about the genes involved in spine formation. A recent study in our laboratory discovered spiny projections on a group of inhibitory neurons called DD neurons in C. elegans. Using C. elegans as a model system, the goal of this project is to identify genes responsible for dendritic spine formation. Specifically, I will 1) conduct a forward genetic screen and examine candidate axon guidance molecules to identify genes involved in dendritic spine formation, and 2) examine the effect hyper- and hypo-presynaptic activity has on the development of spines using locomotory changes, such as thrashing and microfluidic assays, and cell-specific changes using channel rhodopsin and temperature sensitive alleles. Given the strong homology between C. elegans and mammalian genes, this research will help identify conserved mechanisms underlying spine formation and maintenance. Ultimately, understanding spine development will help shape the treatment and prevention of
Microfluidic chips allow for the manipulation, at small quantities of neuronal cells. Further, these chips allow for precise temporal and spatial control. This model is one that can be useful in the framework of neurodegeneration. Deleglise et al (2013) fabricated a 3 chamber microfluidic chip. The chambers held one of: coritical neuron soma and dendrites, cortical axons, or striatal neurons. The chip was designed to replicate an oriented neuronal connection from the coritcal to the striatal cells. Through the use of a chemically induced axotomy, by adding fluid to the central chamber (housing the cortical axon), showed that their chip could be used to simulate a lesion in the neuronal network. An emphasis was placed on the protection of synapses, and event that in axotomy studies, traumatic injury and in many neurodegenerative disorders precedes the loss of neuronal cell soma and axons. They then showed that zVAD-fmk, a caspase inhibitor and resveratrol did not show synaptic protection, while NAD+ and Y27632, a Rho Kinase inhibitor showed significant synaptic protection, despite the mechanism not being clear. Pointing at potential therapeutic targets for neurodegeneration. This can also be further looked at from the functionality of the chip - it is a useful tool in the evaluation of drugs in the axotomy model they presented.
It is important to know that optogenetic silencers can have different effects on synaptic transmission. For example, a light-driven inward Cl-pump, NpHR, causes changes in the reversal potential of the membrane potential of GABA receptors causing a spike after illumination. Arch, also an inward Cl- pump, on the other hand does not result in a spike after illumination. This article provides useful insight of light-activated proteins that can be used as modulators. The ability to change GABA membrane potential can be extremely useful when working with Parkinson models since these receptors are involved in some of the Parkinson’s symptoms.
Precise control over the routes taken by growing neurons is tightly controlled (Steward, 1989). In the brain extracellular cues play an important part prompting attractive or repulsive behaviours in cell migration. In addition, they are necessary for cell adhesion, axon guidance and branching (Erskine & Herrera, 2007). For neurons that navigate over long distances, axons can be guided by chemoattractants that draw axonal navigation in its direction or chemorepellents that deter axonal growth in its region.
The interaction of this major neurotransmitter with different cells in the brain is dependent on whether the cells express receptors for glutamate on their surfaces, to which they can bind and activate. (Hu, Ondrejcak and Rowan, 2012)
It plays a significant role in many neurological functions, including brain plasticity (4), learning and memory (5), and induction of pain (6). Poor or excess release of glutamate can cause serious neurodevelopmental and neurodegenerative disorders such as autism (7-9), epilepsy (7, 10-13), schizophrenia (14), Alzheimer’s disease (14-17) and Parkinson’s disease (18, 19). Physiologically, glutamate is released from pre-synaptic vesicles into neuronal synapses in response to action potential. The concentration of glutamate within the vesicles is reported to be ~100 mmol/L (20). The action potential firing mechanism primarily involves the movement of cations (Ca2+, Na+ and K+) across the neuronal membrane. Two voltage-gated ion-channels located in the axon permit active transport of K+ and Na+, into and out of the cells (20). This ion exchange creates a potential difference of -70 mV (resting potential) through the neuronal membrane. Sudden depolarisation or action potential occurs when potential drops, allowing Na+ to flood into the cells. Action potential is terminated to restore the resting potential of the membrane. Ca2+ channels open to allow the transport of Ca2+ (2). This ultimately causes glutamate to be released from its vesicle into the area between presynaptic membrane and postsynaptic membrane of the subsequent neuron. Through this electrochemical signal
Robert Malenka is a professor in psychiatry and behavioral sciences. Professor Malenka’s laboratory mainly conducts work in two areas of research: the importance of long-lasting activity-dependent changes in the efficacy of synaptic transmission to the development of neural circuits, and the effects of drug abuse to synaptic actions. The first area of research focuses on the understanding of molecular events that lead to synaptic plasticity and changes in synaptic efficacy. Professor Malenka’s laboratory induces dominant negative mutations in different synaptic proteins to study their functions and aims to understand their effect on synaptic plasticity using cellular
This synaptic specificity does not only take place in the cellular layers but also in the subcellular level. Therefore, the signal pathway that occurs in both the cellular and subcellular passes through the positive and the inhibitory cues. The positive cues allow the recognition of partner cells and chaining membranes, however, it can also miscommunicate the recognition to non-target cells. The inhibitory cues are on the non-target cells, and they are essential regulators for synapse specificity. Essentially, these cues function as passages that allow or prevent connections and recognitions of cells, and also help in the distribution of synapses alongside the
Within the hippocampus, long-term potentiation (LTP) results due to the increased activity between a presynaptic and a postsynaptic neuron. The stimulation and subsequent depolarisation of a presynaptic CA3 pyramidal neuron results in the opening of voltage-gated calcium channels (VGCC). Thus, an influx in Ca2+ occurs, and glutamate vesicles are fused via synaptotagmin. The fusing of these vesicles allows the release of glutamate, a neurotransmitter which will bind to AMPA receptors and induce the influx of Na+ into the postsynaptic CA1 pyramidal neuron. This will result in the depolarisation of the postsynaptic neuron, which is one of the requirements for the opening and consequent induction of Ca2+ through the NMDA receptor. Through a cascade
One field of study in the realm of biology that still leaves much to be discovered is neuroscience, more specifically, the neurological pathways and signaling within the brain. While there have been many developments and breakthroughs in identifying areas of activity and neural pathways, there are still many obstacles to overcome and discoveries to be made. The authors of the journal High-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor sought to overcome one such obstacle. They understood the potential for fluorescent proteins to be used for visualizing the firing of action potentials throughout neuronal pathways of the brain, but realized that such proteins were unable to accurately record higher frequency signaling across synapses where there was frequent action potential activity. Within this journal, they describe the methods used to produce a new fluorescent protein, accelerated sensor of action potential 1(ASAP1), and the benefits that it can provide to the field of visualizing the activity of action potentials as they propagate through synapses in the brain. They hypothesized that ASAP1 should be able to give definitive fluorescent readings for the activity of action potentials propagating through the brain, even in areas with high frequency activity that have not been measurable before.
The increase in concentration of calcium is directed by the sarcolemmal calcium current, which is amplified by the SR through a mechanism known as calcium-induced calcium release. To better understand the regulation that goes into this system, Cheng et al. (1993) used fluorescent tagging and confocal microscopes to investigate
A synapse is the space located between the motor neuron and the skeletal muscle which is also referred to as a neuromuscular junction. The motor neurons that originate from the spinal cord, help activate the skeletal muscle fibers. The innervation happens by the processes of the axon. The synapses are present along with these processes and are also known as motor endplate. The neuromuscular junction synapse has three characteristics. It consists of two membranes called the pre and post synaptic membranes and the space between the two membranes is known as the synaptic cleft. A high density of small spherical vesicles are present and they contain neurotransmitter substances. A thickened post synaptic membranes is present and it contains a high