Identity of synaptic vesicles fusing spontaneously

1. Introduction

1.1. Neuro-communication

Signals go through chemical synapses in the central nervous system (CNS) to facilitate neural

communication. The pre-and post-synaptic compartments form a chemical synapse with the

synaptic cleft separating them (Figure 1.1A, B). Voltage-gated Ca2+ channels open as an

incoming electrical impulse leads to Ca2+ influx into the presynapse and results in the

exocytosis of synaptic vesicles, which contain neurotransmitters (NT). Neural tubes release

NTs into the synaptic cleft, where they bind to specific channels, depending on the

neurotransmitter in question, inducing either an excitatory or inhibitory response in the post-

synaptic plasma membrane (Thanapitak, and Toumazou, 2012).

Figure 1.1: Chemical Synapses. Before
an action potential is initiated, a person's muscles are in the resting
state, shown in Figure A. The nerve impulse that enters the presynaptic neuron in diagram B sets off a
chain of seven processes, the first of which is diagram A. At step 1, the neurotransmitter molecules
are contained within membrane vesicles at the presynaptic terminal, where they are then concentrated
and localized. At the 2nd step, the presynaptic membrane depolarizes, primarily as a result of an
impulse from a nerve, but some synapses will only function if the Vm (membrane potential) is
gradually altered. At the 3rd stage, voltage-gated Ca2+ channels open in the terminal, allowing Ca2+
ions to enter. In the following step, an increase in the level of intracellular Ca2+ is responsible for
vesicle fusion with the presynaptic membrane, resulting in a ten million-fold increase in
neurotransmitter release. Ca2+ dependence is provided by synaptotagmins, neuron-specific protein
components of the fusion apparatus, which act as Ca2+ sensors. It only takes one millisecond for each
exocytosis to be completed, thus the fusion events are quick. For step 5, once it has been injected into
the extracellular space, the transmitter is released in fixed quantities and passively diffuses across the
synaptic cleft. The process occurs as follows: at the end of the seventh step, transmitter molecules
leave the receptors and are cleared away via diffusion, enzymatic breakdown, or active absorption
into cells. Additionally, the presynaptic machinery may endocytose the membrane of the exocytosed
synaptic vesicle from the extracellular environment (Boron & Boulpaep, 2016).

1.2. The presynaptic morphology

An SV-containing presynaptic bouton on the hippocampal neurons has a mean of 100-200

SVs. SVs are tiny organelles, which store NTs, that have a diameter of 40 nm. SV exocytosis,
which is Ca2+-dependent, occurs in the active synaptic zone. The active zone is composed of

cell adhesion molecules (including extracellular matrix proteins that affect Ca2+ channel

recruitment), as well as regulatory molecules involved in the fusion of SVs (e.g., as reviewed

in (Südhof, 2012)). Typical electron micrographs of hippocampal boutons show

approximately 5-10 SVs attached to the AZ. (Jahn and Fasshauer, 2012) describe these SVs,

which are primed and docked, as being ready to be deployed at about 100 picoseconds (i.e.,

after receiving an AP (such as is reviewed in (Jahn and Fasshauer, 2012) and are labeled as

the release-ready pool (RRP). Additional classification methods that go beyond

morphological characteristics use functional characteristics. In the example above, the

reserve pool of SVs resists release even when stimulated, as in the case of RRP depletion.

Endocytosis of SVs is observed in the periactive zone outside of the AZ (peri-AZ). These SV

proteins, referred to as the readily retrievable pool, are found in the peri-AZ patches of SV

proteins (RRetP) (Hua et al., 2011). RRP is embedded inside the endocytic vesicle of the cell,

and this tells us that it is approximately the same size as the general endocytic vesicle size

(Wienisch & Klingauf, 2006).

Both the consumer and the manufacturer can use spontaneous or induced release.

According to Peled et al (2014), in Drosophila neuromuscular connections,

spontaneous and induced release can take place at diverse synapses (Melom et al., 2013).

Atasoy et al posit that NMDA receptor populations in the hippocampus are activated by

spontaneous and induced release (2008). When vesicle release was triggered, it was found

concentrated around ribbons, whereas when it was spontaneous, it happened elsewhere

(Zenisek, 2008). When Ca2+ channels were blocked with Cd2+, there was a significant

increase in rod release events around ribbons, but the release was detected in local and distant

places. The non-ribbon release was more common in Ca2+-independent spontaneous release

than induced release (Chen et al., 2013). Ca2+-independent release occurs in the absence of
Ca2+, whereas Ca2+-dependent release occurs only when Ca2+ is present. Nachman-

Clewner et al. (1999) discovered that Ca2+ channels are abundant surrounding ribbons;

therefore, spontaneous release in this environment is likely to involve more ribbon-related

activities than Ca2+-independent activities.

The rod input has reported the ca2+-independent spontaneous release, but the cone

input has not been discovered. Snellman et al (2011) contend that the evoked release is only

likely to occur at the conic ribbons, while Van Hook & Thoreson (2015) agree that the

spontaneous release may occur at the ribbons. The study's findings show that spontaneous

mEPSCs caused by bipolar cell discharge have reduced amplitude and frequency (Mehta et

al., 2013). In RIBEYE mutant mice, the loss of ribbons did not affect bipolar cells rod

spontaneous release (Maxeiner et al., 2016). An alteration in non-ribbon release events

probability may quicken changes in presynaptic transporter current. Thus, researchers have

found no discernible variations in the HC mEPSCs waveform after pulling down the Ca2+

influx. Glutamate levels in the invaginating ribbon synapse rapidly grow as one moves away

from the release point, exceeding the EC50 for glutamate on HCs (Gaal et al., 1998). The

location of the release site may affect the dynamics of HC mEPSC. This is since glutamate

affinity ranges between 1 and 560 lM in heterologous AMPA receptors. (2010) (Traynelis et

al.). The responsiveness of glutamate receptors is diminished, lowering the possibility of

unknown events influencing HC membrane potential. Sensitive receptors, on the other hand,

aid in amplifying the impact.

Sara et al. (2005) found evidence for pool separation and pool mixing (Fredj & Burrone

(2009). Hua et al (2010), on the other hand found eighty-five percent mobility among the

conic and bipolar cytoplasmic vesicular synapses. Traditional synapses, on the other hand,

have only 5% of their vesicles moveable (Groemer & Klingauf, 2007).

 1.3. The Post-synaptic morphology

The post-synaptic membrane comprises transmitter receptors and many proteins grouped

inside the post-synaptic density, according to Boron and Boulpaep (2016). The post-synaptic

membrane is parallel to the presynaptic membrane, with a tiny synaptic gap (30 nm wide)

replete with extracellular fluid separating them. It is necessary for neurotransmitter molecules

synthesized from the axon terminals to infuse across the gap in order to activate postsynaptic

receptors (Easley-Neal, 2011. The most distinguishing anatomical feature of the post-

synaptic side is the post-synaptic density, a film of aggregates observed under a

stereomicroscope on the cytoplical face of the membrane (see Fig. 1.2). The cluster of

transmitter receptors buried within the post-synaptic membrane is still an essential molecular

characteristic of the post-synaptic side (Easley-Neal, 2011). Antibodies, poisons, and ligands,

which are connected to a visible tag molecule, can be used to identify the sites of the

receptors.

The post-synaptic site is the most dominant (90%) of the CNS excitatory synapses of the

dendritic spine. Spines are ubiquitous, implying that they serve important purposes, yet their

modest size (about 1 m long) makes studying their function extremely challenging (Legname,

2013). Spines appear in various forms and varying dendrite-to-dendrite densities (Fig. 1.3); in

fact, some apical neurons lack spines entirely. Spines' post-synaptic density (as with other

centrosymmetric synapses) comprises over thirty highly concentrated proteins comprising

protein kinases, transmitter receptors, a considerable amount of protein clumps, and

endocytosis and glycolysis-related proteins (Shohami et al., 2014).

 Three vesicle-filled presynaptic terminals establish
Figure 1.2: Cochlear nucleus synapses -electron micrograph.
contact with the same post-synaptic dendritic. Arrows represent post-synaptic density (indicating
active zones). (SOURCE: Webster,and Peters, 1976)

Figure 1.3: Dendritic Spines. A:

Golgi-stained material was used to create drawings of numerous dendrites
in the neocortex. Spines are the multiple protrusions. B: Photon micrograph of a neocortical
axospinous synapse. The synaptic spine (S) is situated on the shaft of the dendrite (D), contacting a
peripheral presynaptic terminal (SOURCE: Peters, 1984)

Several functions for spines have also been hypothesized, as reported by Boron and

Boulpaep (2016). Spines may promote the dendritic formation, which may enable synapses to

grow close. Several different research groups have pursued the hypothesis that spines prevent

other cellular synapses. It is possible to employ isolation (either electrical or chemical) in this

case (Koch and Poggio, 1983). When looking at the spine neck and comparing it to the spinal

cord, we find that the neck is thin and inhibits the transmission of electrons as well as the

flow of chemicals between the spine head and the dendrite shaft. Neurons appear to be able

to take in more synaptic input when the spine and neck have increased electrical resistance.

Large amounts of Ca2+ can enter the post-synaptic cell when some excitatory synapses are

activated. As a result, this Ca2+ may be segregated and may increase to higher levels, or it

may be partitioned and avoid affecting other synapses on the cell (Yuste, 2010). The theory

that dendritic spines may be important to learning and memory is far from proved, but it is a

fascinating idea.

1.4. Spontaneous and Evoked Neurotransmission Activity

When a stimulus causes neurons to fire, the communication they send and receive might

be called evoked or spontaneous, according to Hovarth et al. (2020). Both synchronously and

asynchronously, vesicles are released at many synapses due to conventional action potential-

based signaling (Südhof, 2013). Synaptic vesicle release occurs without the aid of an action

potential. An exciting breakthrough was made by genetic research by Kavalali, 2015, which

found that neurotransmission (which is thought to occur in the absence of prior stimuli) has

been discovered to use partially different molecular equipment to operate at unique post-

synaptic locations while evoked neurotransmission (which is stimulated by prior stimuli) uses

various machinery and has distinct post-synaptic locations.

 The excitatory and inhibitory neurotransmission organizing principles may differ. AMPA

‘(α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)’ receptors activation occurs at the

hippocampal excitatory synapses both at the Drosophila neuromuscular junction and

neuromuscular junction synapses of mammals (Sara et al., 2011; Peled et al., 2014; Melom et

al., 2013). NMDA receptor distribution in the hippocampus is close to ideal due to

spontaneous and triggered glutamate release (Reese and Kavalali, 2016; Atasoy et al., 2008).

In summary, these studies proved that the initiation and stimulation of neurotransmission are

happening at the same synapse. However, only one kind of NMDA and one type of AMPA

receptor activation are taking place simultaneously, according to Crawford et al. (2017),

Gonzalez-Islas et al. (2018), and Sutton et al. (2004), and it is necessary because of the

existence of spontaneous and evoked neurotransmission at excitatory synapses (Fong et al.,

2015; Ramirez et al., 2017; Sutton et al., 2007; Nosyreva et al., 2013; Sutton et al., 2006).

1.5. Synaptic vesicles pools

In agreement with the research done by Hua et al. (2011), synaptic vesicles can be

separated into a recycling pool that is responsible for triggering neurotransmitter release and

a resting pool that is unaffected by stimulation. Additionally, Fredj and Burrone (2009)

argued that recycling pools entail reserve and rapidly released pools.

1.5.1. Recycling pool

Südhof (2004); Soykan, Maritzen, and Haucke (2016); Rizzoli and Betz (2005); and

Denker and Rizzoli (2010) all report that for recurrent release, Vesicle recycling takes place

at the presynaptic terminal, inside the synaptic vesicle (SV). Conceptually recycling SVs

include exocytosis, endocytosis, and intra-terminal trafficking. An effective kinetic

framework supporting sustained consumption and regeneration of SVs is required to maintain

synaptic efficacy (Qiu, Zhu, and Sun, 2015; Mohrmann et al., 2010; and Alabi & Tsien,

2012). Clathrin-independent rapid endocytosis, kiss-and-run, and ADBE (activity-dependent

bulk endocytosis) are several strategies for recycling synaptic vesicles, dependent on how

much activity is being done and the specific synapse type (Watanabe et al., 2014; Smith,

Renden, & Watanabe & Boucrot, 2017).

While SV endocytosis has received the bulk of attention in SV characterization, little

consideration has been devoted to post-endocytic SV trafficking. Presynaptic terminals

contain hundreds to thousands of SVs, and 65% of these SVs are involved in recycling,

which is referred to as the recycling pool (RP) (Qiu et al., 2015; Denker & Rizzoli, 2010). In

RP, there was significant diversity in SVs' geographical distribution and reusability (Qiu et

al., 2015; Denker & Rizzoli, 2010). Aside from the well-defined and tested RRP (readily

releasable pool), While no one has solved the mechanism by which SVs in nerve terminals

are kinetically arranged for synaptic transmission, researchers are still actively searching for a

solution. The existence of a surface reservoir of SV membrane at probable endocytosis sites

is uncertain, although SV membrane may also exist in abundance at nerve terminals, as

evidenced by the SVV/synaptobrevin and synaptotagmin transport properties (Klingauf,

2011; Hua et al., 2011).

According to a recent study, the nerve terminal SVs are produced kinetically from

many pools with varied reuse preferences and timing (Miki et al., 2018). To demonstrate that

vesicle recycling occurred at the calyx of the Held synapse during in vivo-like activities,

researchers at Sun et al. (2021) examined presynaptic capacitance and evoked excitatory

post-synaptic current (EPSC) recordings on the calyx of the Held synapse. The recycling

process is separated into four pools, each of which works as a regulatory phase. Because of

this, the RP's integrated kinetic structure100 could both be a reliable source of SVs and help

to keep synapses from losing connection.

1.6. Resting pool

 The resting vesicle pool or RtP is a group of vesicles that remains unreleased even

after stimulation that induces a saturating degree of vesicular turnover, as reported by Alabi

(2012) and Tsien (2012). Fluorescence microscopy (Fernandez-Alfonso and Ryan 2008;

Poskanzer and Davis 2004) or external uptake of genetically encoded pHluorin probes, as

determined by EM of electron-dense QD cores (Zhang et al. 2007). (Harata et al. 2001). The

estimations of the RtP size distribution might vary greatly; nevertheless, in some cases, it

may be as low as around 50% – 85% of all vesicles (Fernandez-Alfonso and Ryan 2008;

Harata et al. 2001b). De Lange et al. (2003) and NMJs of Drosophila (Poskanzer and Davis

2004) have been proposed to function similarly (Wyatt and Balice-Gordon 2008).

Nonetheless, 15% of vesicles remain labeled even in terminals that are reported to lack an

RtP. (Richards et al. 2003).

Surprisingly, so many vesicles are seen in the RtP while so few SVs are present. A

portion of the rationale is that only a limited number of vesicles are required to keep

neurotransmission running even without RtP activation (Denker et al., 2011a). RtP vesicles

are attracted by intracellular signaling pathways as well. Additionally, the Drosophila

neuromuscular junction (NMJ) vesicles that are the least amenable to exocytosis can be made

to go to the RRP using a protein kinase (Kuromi and Kidokoro 2005). It is possible to prevent

the cyclin-dependent kinase 5 (CDK5) activation in hippocampus terminals by inhibition of

the cyclin-dependent kinase 5 (CDK5) (Kim and Ryan 2010). Prolonged neuronal silence

leads to homeostatic scaling of presynaptic release (Thiagarajan et al., 2005). Restoration of

the RtP requires the calcineurin-dependent phosphatase calcineurin to take an opposing

action. As shown in Fig. 2, opposing effects of phosphorylation and dephosphorylation can

cause synaptic strength to shift and adapt in a way that results in little changes to overall

vesicle number or bouton size (Ratnayaka et al. 2012).

Figure 1.4: A simplified representation of the function and regulation of the vesicle pool (SOURCE: Alabi, Tsien, 2012).
According to research, RRP vesicles are believed to reside within proximity to the active zone for the
highest Pves, leading to a rapid fusion event. The vesicle clusters and the RP and RtP vesicles are
scattered inside them. They both help the membrane vesicle superpond come into being (arrows
leaving bouton). The RtP vesicles include molecular labels and contribute to spontaneous release at
extra-AZ locations (Hua et al., 2011; Ramirez et al., 2012). RRP vesicle shading is a potential
reflection of RRP heterogeneity. By a factor of 5, the sum of vesicles shown in the bouton has been
made small. The column on the right will be highlighted in red if you make any changes to the
number or attributes of the vesicle pool. Every alteration impacts the wiring and flexibility of the
synapses (see text for further details). The NRRP and Pves values may alter with RRP. For RP, an
increase in the size of the bouton can hold more RP (and RtP) vesicles, and as a result, lengthen the
length of the active zone (which could lead to RRP expansion) (Welzel et al. 2011). Furthermore, the
stimulation of calcium or protein kinase C will enhance RP mobilization, reducing RRP depletion.
The CDK5 enzyme is inhibited in RtP, resulting in increased vesicle retrieval to the TRP. An alternate
molecular label is proposed (Ramirez et al., 2012).
 1.7. The spontaneous synaptic vesicle pool has several points of view

When soluble protein Doc2b (beta-protein isoform) is associated with vesicles containing

synaptotagmin 1, these organelles are at risk for both spontaneous and induced release,

claims the report published by Truckenbrodt and Rizzoli (2014). Walter et al. (2011) also

replicate these findings. Alternatively, if just one of these molecules is present in the vesicles,

they only release spontaneously or only when stimulated. Presently, the question of whether

all vesicles have sensor molecules is unknown. The synaptotagmin-1 copy numbers (about 15

synaptotagmins-1 molecules per synaptotagmin-1 vesicle, on average) combined with the

numerous copies of Doc2 (approximately 10 per vesicle, on average) suggest that most of the

authentic synaptic vesicles are associated with at least a few copies of both (Wilhelm et al.,

2014).

Truckenbrodt and Rizzoli (2014) also contend that a dispute about whether spontaneous

release occurs from the same synaptic vesicle pool as that of triggered release has been

compounded by much contradictory research that has been conducted in the last several years

(quoting Wilhelm et al., 2010; Groemer and Klingauf, 2007; Fredj and Burrone, 2009; Sara et

al., 2005; Mathew et al., 2008 as the examples). Also, Truckenbrodt and Rizzoli (2014) argue

that recent studies have deliberated on the role of spontaneous recycling in neuronal cultures

by blocking excitatory neuron activity using the neurotoxin tetrodotoxin (TTX), which

abolishes action potentials.

The researchers, Cork et al. (2016), also investigated the dependence of Ca2+ on vesicle

release at photoreceptor ribbon synapses, the quantity of those vesicle pools, and the

locations where those vesicles spontaneously leak. They contended that each organization is

searching for talented, self-motivated individuals passionate about making a difference in

question about the synaptic pool of the neural activity. Numerous investigations have

demonstrated that spontaneous release necessitates intracellular Ca2+, which provides varied
Ca2+ concentrations. Ion channel entrance by Ca2+ can enhance spontaneous release. Ca2+

chelators, BAPTA, or EGTA can be used to limit spontaneous release according to Schneider

et al. significantly, 2015 in conjunction with the findings from Xu et al. (2009), Goswami et

al., 2012; Ermolyuk et al. (2013). Voltage-gated Ca2+ channels were shut when Ca2+

channels in cortical and cerebellar neurons were cut in half in the studies that Goswami et al.

(2012) and Williams et al. (2012) conducted. In this regard, Cork et al. (2016) found that

most spontaneous release in brainstem neurons was caused by calcium influx through

tonically activated TRPV1 receptors (Peters et al., 2010; Shoudai et al., 2010).

Cork et al. (2016) also discovered that the membrane potential of vertebrate

photoreceptors varies continually with light intensity, allowing for adaptations to

accommodate differences in light intensity and adaptation state. Rod and cone cells are kept

at -40 mV in the dark, causing Ca2+ channels to open and release the cell's contents. Ca2+

influx inhibition, as expected, lowered mEPSC frequency. However, the flow of information

did not stop. Thus, the team discovered spontaneous quantal glutamate transporter currents in

voltage-clamped rod and cone cells in the absence of L-type Ca2+ channels. By inhibiting

Ca2+ channels with Cd2, the frequencies of these currents were lowered instead of being

completely abolished.

Cork et al. observed three distinct types of basal photoreceptor release (2016). Both

spontaneous and induced release remain after Ca2+ input, either Ca2+-free extracellular

fluids or Cd2+ is reduced. The remaining mEPSCs are regulated by a Ca2+-independent

release mechanism, which is also in charge of terminating mEPSCs when Ca2+ input is

inhibited.

Deitcher et al. (1998), for example, feel that there are two independent mechanisms at work,

whether a gene's expression is activated or when it is released on its own. However, Smith et

al. (2012) argue that the exact mechanisms govern expressed and released gene activity. Even
though Ca2+ independence is hypothesized, thermodynamic changes in SNARE

conformation lead to spontaneous release.

Doc2 and other Ca2+ sensors may be essential for Ca2+-dependent spontaneous release

(Groffen et al., 2010). Doc2 may be jeopardized by Ca2+-independent spontaneity (Pang et

al., 2011). In photoreceptor synapses, the exocytosis sensor has high Ca2+ affinity but low

cooperativity (Lou et al., 2005; Sun et al., 2007). Non-canonical SNAREs, such as VAMP7

(Hua et al., 2011) and Vti1a (Mychajliw et al., 2009), can also release spontaneously

(Ramirez et al., 2012). Long or short exposure to different SNARE isoforms or related

proteins may differ in how their activities, influence spontaneous or triggered release,

according to Hua et al. (1998) and Scheuber et al. (2006) (Maximov et al., 2009; Buhl et al.,

2013). According to Weber et al. (2010), "Complexin 3/4" expression at ribbon synapses

decreases spontaneous bipolar cell release." (Vaithianathan et al., 2013; Vaithianathan et al.,

2015) Despite the discovery of a few chemical mediators, there is no obvious mechanism for

spontaneous release.

1.8. Is it the same or a different pool?

Ramirez and Kavalali (2012) conducted a study to understand how non-canonical

SNAREs contribute to vesicle recycle. In their paper, Ramirez and Kavalali argued that a

growing body of evidence suggests that different pools of synaptic vesicles are responsible

for different types of neurotransmission. In their study, Fredj and Burrone (2009) suggested

that therare variably responses to phorbol ester regulation and dynamin inhibition (Chung et

al., 2010). GABAergic terminals release smaller vesicles at rest (Mathew et al., 2008) and

larger numbers with stimulation, found throughout neuronal development and synaptic

maturation. (Andrew et al 2012; Mozhayeva et al., 2002). Furthermore, the vesicle fusion rate

may differ according to the kind of neurotransmission used (De'k et al., 2006; Weber et al.,

2010). While numerous research has established that spontaneous versus evoked release was
linked to a similar vesicle pool, Ramirez and Kavalali (2012) still contend that this notion is

controversial since many of these investigations dispute the hypothesis behind the pool of the

vesicles (Groemer & Klingauf, 2007; Hua et al., 2010; Wilhelm et al., 2010). Past genetic

knockout investigations imply that a small number of vesicles drive spontaneous and induced

synaptic vesicle fusion that deviate from the SNARE composition, so some percentage of

vesicles contributing to both activities may be distinct pools. Asynchronous and spontaneous

neurotransmitter release was detected by Ramirez and Kavalali (2012) and induced release

using molecular tags for vesicles produced during various types of neurotransmission. The

duo then presented the SNARE proteins, as well as a variety of other non-canonical SNARE

proteins. Nonetheless, this study focussed much on the SNARE proteins.

1.8.1. SNARE Proteins

The SNARE family of proteins is crucial for organelle communication, as Dingjan et

al. (2018) suggested. SNAREs are necessary for cell membrane fusion, too. The results of

this study illustrate that membrane fusion is a major process for all living species, which

influences a wide range of biological activities, including the replication of viruses, cell

fertilization, and the movement of cellular substances into and out of cells among others.

However, this happens because the SNARE molecules in both the benefactor and destination

organelle membranes have already been complexed (Dingjan et al., 2018). The compartments

of eukaryotic cells, especially those that house several membrane-enclosed systems, must

interchange their contents and exchange information across membranes. SNARE proteins,

which are important components of the eukaryotic fusion machinery responsible for fusing

synaptic vesicles with plasma membrane, are proven to cooperate to effect efficient and

regulated fusion. Synaptobrevin (endocytosis) and syntaxin (target fibroblast) work together

to form a core trans-SNARE complex during exocytosis. When several stages of exocytosis

are included, the complex plays a diverse range of roles during the process of priming, fusion
pore development, and enlargement until, ultimately, vesicle release or exchange is

completed (Han et al., 2017).

Although SNARE proteins transport molecules between endosomes and phagosomes via

the endocytic route, SNARE proteins also play a role in trafficking molecules between

different organelles, such as lysosomes, the Golgi apparatus, the plasma membrane, and the

endoplasmic reticulum. An in-depth explanation of the SNAREs involved in endosomal and

phagosomal trafficking was provided by Dingjan et al. (2018). In humans, out of the 38

SNAREs, 30 are located at endosomes or phagosomes. To circumvent nutritional breakdown,

infections often target SNARE proteins and reroute intracellular transport to allow them to

acquire access to nutrients, escape recognition by the immune system, and inhibit nutrient

breakdown. A complicated transport network consisting of numerous SNAREs is helping to

describe a complicated trafficking pattern.

According to Weber et al., SNARE complexes combine to initiate signal transduction by

compressing membranes together (1998). At the heart of SNARE complex formations is a

'zero layer' composed of a hydrophilic electrostatic connection mediated by three glutamine

residues and one arginine residue (Sutton et al., 1998). SNARE proteins are classified as Q-

SNAREs (such as syntaxin-1 and SNAP-25) or R-SNAREs (such as syb2) based on the

pattern of their SNARE sequences (Fasshauer,1998). All R-SNAREs have a SNARE motif

accompanied by a transmembrane anchor. A set of R-SNAREs is distinguished by either a

small N-terminal region at the start of the SNARE motif, known as the brevins, or an

extended N-terminal portion that is more than 120 to 140 amino acids long, known as the

longins (Rossi et al., 2004). The major synaptic vesicle R-SNARE, syb2/VAMP2, and the

prototype VAMP7, which is present in some synaptic terminals, are examples of the longin

Brevin subclass of vesicular (v-) SNAREs (Scheuber et al., 2006). Using mass spectrometry,

R-SNAREs such as syb1/VAMP1, cellubrevin/VAMP3, VAMP4, and the structurally related

Q-SNARE Vti1a (Grnborg et al., 2010) are found in synaptic vesicles. Vti1a-bound SNAREs

have recently been shown to promote fusion in vitro at levels comparable to Syb2 (Hung et

al., 2010). This establishes the different roles of these proteins in neurotransmitter release.

1.8.2. Vti1a SNARE Protein

Vti1a, another synaptic vesicle SNARE, stimulates spontaneous neurotransmission,

which seems to play a particular role (Ramirez et al., 2012). As far as researchers could tell,

in the trials using optical imaging, vesicles containing pHluorin-tagged vti1a did not

mobilize, but this did not apply to inactivity. Inhibitory and excitatory synapses exhibited

bidirectional spontaneous event frequency effects in the absence of impacts on evoked

neurotransmission, as predicted by the electrophysiological effect. These findings are

consistent with recent proteomic study, which found no difference in vti1a expression on

excitatory and inhibitory synaptic vesicles (Grnborg et al., 2010). Furthermore, deletion of

vti1a can facilitate spontaneous neurotransmission in the absence of the traditional v-

SNARE, syb2, as revealed in syb2 knockout neurons. The findings, when combined with

optical observations of concurrent evoked syb2 release with low VTI1A response in the same

boutons, support the hypothesis that neurotransmitter vesicle pools can be separated based on

whether or not spontaneous or evoked release is occurring (fig 1.5).

 S YN2, which is located at the
Figure 1.5: A model for the distribution of syb2, VAMP7 VTI1A vesicular SNAREs.
intersections of neuronal membranes, serves as the principal vesicular SNARE to guarantee that
synaptic connections are formed promptly. A non-canonical SNARE pathway is implicated in vesicle
fusion and recycling in mice that have had the Syb2 gene deleted. According to recent investigations,
both vti1a and VAMP7 may be capable of fulfilling this job and traffic specifically at rest. In the
absence of activity, Vti1a has a larger intracellular pool and more robust trafficking. The alternative
interpretation is that syb2-carrying vesicles are more sluggish to respond to action potentials
generated stimulation, while vesicles containing VAMP7 and vti1a are reluctant to follow the action
potential. Given their considerable aversion for mobilization, the percentage of vesicles dominating
the resting pool, which is made up of VAMP7-containing vesicles, could be somewhere between zero
and one percent. In the case of regulatory circuits that influence various neurotransmission types,
diverse cellular responses may be triggered due to the differing fusion properties of distinct
populations of vesicular synaptic vesicles. If the presynaptic activity is comparatively great, the
consequent post-synaptic signaling activities will have a greater influence.

1.9. The aim of the study

i) In light of the above studies, there is still confusion about whether or not the

spontaneous and evoked neurotransmissions are mobilized from the same pool or

not. Therefore, this study purposed to shed light on the confusion that exists from

the previous studies and once and for all come up with the most accurate answer
 regarding the pools of the neurotransmission activities-whether they occur from

one pool or separate ones.

ii) Opazo et al (2010) previously completed a study, and this new study continues the

examination begun by them. Using antibody labeling, Opazo et colleagues

discovered that numerous synaptic vesicle markers are grouped on the plasma

membrane (although GFP-tagged variants were not). Like other synaptotagmin

proteins, the team found that the native synaptotagmin needed large amounts of

stimulation to diffuse from synapses, but it appeared to recycle at lower amounts of

activation. To add, there was no intermixing of native synaptotagmin molecules

when recycled plaques were tested in the presence of distinct labels. In light of the

discoveries mentioned above, we are excited to test the hypothesis that two

separate vesicle pools can mix utilizing a novel optogenetic technique known as

Phoenix and an experimentally demonstrated glutamate sensor (iGluSnFR)

(Marvin et al., 2013; Marvin et al., 2018).

Below are the specific objectives of the study:

iii) To identify the pool for spontaneous neurotransmission activity

iv) To identify the pool for evoked neurotransmission activity

v) To identify the similarity and differences of the spontaneous and evoked

neurotransmission activities;
 2.0. Materials and Methods
2.1. Reagents

3.0. References