Article

ISSN 0006-2979, Biochemistry (Moscow), 2026, Vol. 91, No. 4, pp. 561-587 © Pleiades Publishing, Ltd., 2026.

561

REVIEW

Functioning of Synaptic Vesicle Pools:

Diversity and Organizational Principles

Chulpan R. Gafurova

1,a

* and Alexey M. Petrov

1,2

Kazan Institute of Biochemistry and Biophysics,

Kazan Scientific Center of the Russian Academy of Sciences, 420111 Kazan, Russia

Kazan State Medical University, 420012 Kazan, Russia

e-mail: gafurova7090@gmail.com

Received February 25, 2026

Revised March 26, 2026

Accepted March 26, 2026

Abstract— Presynaptic nerve terminals contain a large number of vesicles filled with neurotransmitters,

whose release ensures signal transmission from the presynaptic neuron to the postsynaptic cell. Despite

their morphological homogeneity, synaptic vesicles (SVs) are functionally heterogeneous and are organized

into distinct groups (pools) that differ in their ability for exocytosis and mobilization, recycling kinetics, and

protein composition. In addition to the classic pools – the readily releasable pool (RRP), recycling pool, and

reserve pool – other populations have been identified, including spontaneously recycling vesicles, vesicles

of resting pool and superpool. Vesicles from different pools engage in different modes of exocytosis and

endocytosis, and the extent of interpool mixing varies depending on the synapse type and physiological

or pathological conditions. Changes in the organization of SV pools underlie multiple forms of synaptic

plasticity. Furthermore, SV cycling is a target of several pharmacological agents, and its disruption plays

a significant role in the pathogenesis of neurodegenerative diseases. This article is a systematic review of

SV pools, their organizational features in central and peripheral synapses, and implications of changes in

the structure of SV pools in synaptic plasticity, action of drugs, and development of neurological disorders.

DOI: 10.1134/S0006297926600535

Keywords: active zone, vesicle recycling, synaptic vesicle, presynaptic nerve terminal, synaptic vesicle pool,

exocytosis, endocytosis

* To whom correspondence should be addressed.

INTRODUCTION

In the nervous system, signal transmission from

a presynaptic cell to a postsynaptic cell occurs pri-

marily at chemical synapses, where the two cells are

separated by a synaptic cleft. This process typically

takes a fraction of millisecond to several millisec-

onds and proceeds via release of neurotransmitters

from the presynaptic terminal and their subsequent

binding to receptors on the postsynaptic membrane.

In the presynaptic terminal, neurotransmitter mole-

cules are stored in 40 to 50-nm synaptic vesicles (SVs).

SV cycling is a fundamental mechanism underly-

ing neurotransmission. In response to the action po-

tential, vesicles undergo exocytosis, resulting in rapid

(less than 1 ms) release of neurotransmitters into the

synaptic cleft. Vesicle endocytosis and reloading with

neurotransmitter molecules occur within tens of sec-

onds or even minutes and replenish the pool of SVs

available for subsequent rounds of exocytosis [1].

Despite being morphologically similar, SVs are

functionally and biochemically heterogeneous and

can be classified into distinct populations (pools).

The general vesicle pools include the readily releas-

able pool (RRP), which is the first to undergo exocyto-

sis; the recycling pool, which sustains neurotransmis-

sion during moderate activity; and the reserve pool,

which is mobilized during periods of intense synaptic

activity [2]. Recent studies have identified more spe-

cialized vesicle populations, including spontaneously

recycling vesicles[3], the resting pool, which does not

normally participate in neurotransmitter release [4],

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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 4 2026

and the “superpool” containing the vesicles capable

of exchanging between synaptic boutons [5].

Some pools can overlap; for example, vesicles

from the reserve pool may transit to the superpool

and vice versa [5]. Other pools are less prone to in-

termix and retain their identity even after multiple

rounds of exo- and endocytosis. SVs from different

pools exhibit functional heterogeneity, as they can

release neurotransmitter and then be recovered via

different modes of exocytosis (synchronous, asynchro-

nous, spontaneous) and endocytosis (clathrin-depen-

dent, kiss-and-run, ultrafast, and bulk), respectively.

Vesicles from different pools may also differ in mo-

lecular composition, which likely contributes to the

functional specialization of vesicle populations [6].

The organization of vesicle pools varies across

synapses, reflecting adaptations to specific patterns of

synaptic activity. Changes in the structure of SV pools

underlie synaptic plasticity. Moreover, SV cycling is

a target for various pharmacological agents, such as

fluoxetine [7] and atorvastatin [8]. Disruptions in the

vesicle cycling are implicated in the pathogenesis

of neurological disorders, such as Alzheimer’s dis-

ease [9], Parkinson’s disease [10, 11], schizophrenia

[12], and amyotrophic lateral sclerosis (ALS) [13, 14].

This article discusses the known SV pools, their

properties and functional features, and the modes of

exo- and endocytosis associated with each pool, as

well as examines the features of SV pool organization

in various systems, such as hippocampal synapses,

calyx of Held synapses, Drosophila, frog and mouse

neuromuscular junctions (NMJs), and ribbon synaps-

es. We also addressed changes in the organization of

SV pools during the induction of synaptic plasticity,

under the action of biologically active substances,

and in neurological disorders.

READILY RELEASABLE POOL (RRP)

OF SYNAPTIC VESICLES

The RRP comprises a small fraction of SVs (typ-

ically 1-2% of the total). Vesicles of this pool are the

first to undergo exocytosis upon the presynaptic ter-

minal activation (Fig.1). Most RRP vesicles are docked

at the presynaptic membrane in a specialized region

adapted for exocytosis, known as the active zone (AZ)

[2, 15-18]. SNARE proteins in these vesicles and in

the AZ are partially coiled and are in a fusion-ready

(primed) state [19]. However, docking does not neces-

sarily imply priming: not all docked vesicles belong

to the RRP. For example, in cultured hippocampal

neurons, the number of RRP vesicles is lower than

the number of docked vesicles [15]. Conversely, not

all RRP vesicles are docked. Thus, frog NMJs contain

a subset of RRP vesicles in the cytoplasm of the nerve

terminal [20], suggesting the existence of an ultrafast

mechanism that rapidly recruits these SVs to sites of

exocytosis.

In the central nervous system (CNS), stimulation

of a single synapse often fails to trigger the neu-

rotransmitter release. In this case, the probability of

evoked vesicle exocytosis at the nerve terminal de-

pends on both the RRP size and the release probabili-

ty of an individual vesicle within RRP[21]. Therefore,

modulation of the RRP size plays an important role

in the formation of synaptic plasticity and regulation

of synaptic strength.

SVs in the RRP are not functionally uniform;

rather, the pool exhibits heterogeneity (Fig.  1). Stud-

ies conducted in mouse NMJs have shown that

during high-frequency stimulation, the number of

RRP vesicles is roughly equivalent to the number of

docked SVs. In contrast, during low-frequency stim-

ulation, the size of the RRP is substantially smaller

and represents only a fraction of the docked vesicle

population. This suggests that in mouse NMJs, some

docked vesicles may remain functionally silent during

low-frequency stimulation, but are released as a part

of the RRP during high-frequency stimulation, ensur-

ing more efficient synaptic transmission under these

conditions. One possible explanation for this fact is

that not all AZs are engaged during the low-frequen-

cy stimulation. Certain AZs may form clusters (mul-

timeric AZs) characterized by a high density of Ca

channels and, consequently, a higher probability of

SV release. Under low-frequency stimulation, neu-

rotransmitter release may occur from these multim-

eric AZs, while unitary AZs remain dormant and are

recruited only during high-frequency stimulation[22].

Alternatively, heterogeneity may arise from differenc-

es in the vesicle molecular composition and intrinsic

sensitivity [23]. Consistent with this view, matu-

ration is associated with an increase in the RRP size

in mouse NMJs, accompanied by a more diffuse dis-

tribution of SVs within the axoplasm [24].

In calyx of Held synapses, neurotransmitter re-

lease from the RRP exhibits two components – fast

and slow – reflecting the presence of two vesicle

subpopulations with different sensitivity to cyto-

solic Ca

 [25]. This heterogeneity contributes to

the short-term plasticity: the fraction of rapidly re-

leasable vesicles increases after tetanic stimulation,

thereby increasing the number of released vesicles at

the onset of subsequent stimulation [26].

A similar vesicle heterogeneity is also observed

in synapses formed between granule cells and

Purkinje cells in the cerebellum. The presynaptic

terminals of granule cells contain two SV subpop-

ulations in the RRP. One of them is released in re-

sponse to single stimuli, but ceases to be released

during the following low-frequency stimulation, likely

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Fig.  1. The functioning of SVs of the RRP depends on its organization and replenishment mechanisms. Most RRP vesicles

are primed in the AZ, while a smaller fraction resides away from the membrane but can rapidly dock at exocytosis sites

in response to neuronal activation. Certain regions of the presynaptic terminal may also serve as precursors for the

formation of new exocytosis sites during synaptic plasticity induction. Some RRP vesicles release neurotransmitter in re-

sponse to low-frequency stimulation and may also undergo spontaneous fusion, contributing to spontaneous exocytosis.

Other RRP vesicles are preferentially engaged during high-frequency stimulation, reflecting differences in both vesicle

properties and characteristics of associated exocytosis sites. RRP replenishment can occur through the mobilization of

vesicles from other pools (e.g., recycling and reserve pools) and recycling by endocytosis. The prevalent endocytosis mech-

anism for RRP replenishment can vary across different synapse types. Clathrin-mediated endocytosis (CME), which is a

relatively slow process, primarily sustains vesicle supply during low-frequency activation, while ultrafast endocytosis

(UFE) followed by endosomal sorting enables rapid recovery of vesicles after brief bursts of high-frequency stimulation.

Additional mechanisms may also contribute to vesicle recycling, such as kiss-and-run exocytosis, in which vesicles tran-

siently fuse and rapidly detach without full collapse into the presynaptic membrane. Following endocytosis, vesicles can

be redistributed to another pool, targeted for degradation, or transported to the soma for signaling functions. Dynamic

exchange between vesicle pools also takes place.

acting as a filter for weak or “insignificant” stimuli.

The second subpopulation is recruited only during

the high-frequency stimulation and remains large-

ly inactive at low frequencies. This RRP fraction is

characterized by faster recycling, ensuring more ef-

ficient neurotransmission during high-frequency ac-

tivity [27, 28].

The mechanisms underlying RRP heterogeneity

remain poorly understood. Contributing factors like-

ly include the number of voltage-gated Ca

channels

at individual exocytosis sites and their properties

(post-translational modifications, interaction with

partner proteins, subunit composition), location of

docked synaptic vesicles relative to Ca

channels

[29-31], different extent of vesicle priming in the

AZ (e.g., superpriming  [32]), the properties of exo-

cytosis sites themselves, protein and lipid composi-

tion of SVs [33], and local conditions, such as nearby

spontaneous exocytosis events, random opening of

or K

channels, etc. The factors determining SV

heterogeneity likely vary in different synapses, have

their own properties, and dynamically adjust in re-

sponse to ongoing patterns of synaptic activity.

Changes in the RRP structure. Changes in the

number of SVs, as well in the proportion of active

SVs in the RRP, play an important role in synaptic

plasticity. For example, in hippocampal neurons, ac-

tivation of cAMP-dependent protein kinase A path-

way, which induces long-term potentiation (LTP),

leads to the increase in the number of docked SVs

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in the AZ  [34] together with remodeling of the AZ

ultrastructure, when docked “non-releasable” SVs

from the reserve pool are replaced with active RRP

vesicles [35]. High-frequency stimulation, as well as

activation of the Sema3a–PlexinA4–ITGB1 pathway in-

volved in presynaptic plasticity, cause an increase in

the number of RRP vesicles by mobilizing SVs from

the reserve pool [35, 36]. Expansion of the RRP after

LTP induction increases the number of SVs available

for release, thereby improving the reliability of syn-

aptic transmission, and is necessary for the formation

of stronger connections [35]. Activation of GABA

re-

ceptors in the nerve terminals of the medial habenu-

la causes a 4-fold increase in the RRP size and the

number of docked vesicles due to the translocation

of CAPS2 proteins (Ca

-dependent activator protein

for secretion 2) associated with additional vesicles to

the AZ within a few minutes [37]. Ca

-dependent ac-

cumulation of SVs in the RRP has been implicated in

the short-term facilitation of neurotransmitter release

during paired stimulation in Drosophila NMJs [38].

The opposite scenario is also possible: vesicles

can leave the RRP and localize within 100  nm of

the AZ. These SVs can reoccupy free exocytosis sites,

although they may alternatively relocate to the re-

serve pool [39]. Some SVs can undergo rapid (with-

in 100  ms) undocking from the AZ. Such undocking

events are several times more frequent than spon-

taneous exocytosis [40]. In hippocampal synapses,

stimulation can trigger the undocking of certain ves-

icles from the presynaptic membrane instead of fu-

sion [41]. In some types of central synapses, repeated

low-frequency stimulation leads to synaptic depres-

sion. One possible mechanism underlying this effect

is reduction in the RRP size driven by vesicle undock-

ing and transition to another pool [42].

Disruptions in the RRP structure are observed in

certain pathologies. For example, in epilepsy, inhibi-

tory synapses show an increased number of docked

SVs, while the proportion of fusion-ready (primed)

SVs among them decreases [43], indicating a reduc-

tion in the RRP size.

RRP replenishment. The RRP can be replen-

ished through multiple pathways, including recruit-

ment from reserve and recycling pools, as well as

endocytosis (Fig.  1). In large synapses, such as calyx

of Held synapses, RRP replenishment occurs via ves-

icle recruitment from the recycling or reserve pool,

providing their continuous supply to the AZ [44-46].

Synapses between parallel fibers and interneurons of

the cerebellar molecular layer contain the so-called

intermediate, or upstream, pool. Positioned function-

ally between the recycling pool and the RRP (i.e., up-

stream of the RRP), this pool contains a small number

of SVs (1-4 vesicles per release site or 6-20 vesicles

per AZ) and serves as a source for RRP replenish-

ment. It is quickly depleted (within ~10 action poten-

tials) and is restored within 0.5-1  s, likely via vesicle

recruitment from the recycling pool. Such replenish-

ment dynamics is essential for maintaining reliable

responses to temporally separated bursts of stimuli

[47]. Furthermore, the intermediate pool is recovered

through a fast, dynamin-dependent endocytotic path-

way that operates on a timescale of ~10  s and can

generate up to ~200 vesicles per AZ, thus preventing

excessive depletion of the recycling pool [48].

In hippocampal synapses, the RRP can be main-

tain by recycling for a relatively long time without

SV mobilization from the reserve pool, i.e., RRP re-

plenishment occurs through endocytosis  [49]. One

possible explanation is that these synapses contain a

relatively small number of vesicles, so the efficien-

cy of the existing endocytic machinery is sufficient

for rapid and efficient SV recycling. Furthermore, the

mechanism of endocytosis varies between the syn-

apse types. In inhibitory hippocampal synapses, the

kiss-and-run endocytosis predominates, whereas ex-

citatory synapses primarily rely on clathrin-mediated

endocytosis [50]. Interestingly, in postganglionic sym-

pathetic nerve terminals, exocytosis of RRP vesicles is

coupled with a mechanism ensuring rapid reuptake

of catecholamines into the presynaptic terminal for

RRP vesicle refilling [51].

The mechanisms by which endocytosis replen-

ishes the RRP vary depending on the synapse type

and physiological conditions, including patterns of

neuronal activity. In presynaptic terminals, the pre-

dominant pathway is clathrin-mediated endocytosis.

Following vesicle fusion with the presynaptic mem-

brane, vesicular components are redistributed to

endocytosis sites (peri-AZs), where vesicles bud off

via a clathrin-dependent process. In cortical and hip-

pocampal synapses, as well as in cultured neurons,

released vesicles can be retrieved via clathrin-medi-

ated endocytosis, thus replenishing the RRP [52, 53].

Clathrin-mediated endocytosis operates efficiently

during moderate levels of synaptic activity. In con-

trast, at higher stimulation frequencies, faster endo-

cytotic mechanisms, such as kiss-and-run and ultra-

fast endocytosis, predominate, enabling more rapid

recycling of RRP vesicles [54-56].

In the kiss-and-run mechanism, neurotransmitter

molecules are released from the vesicles through a

small protein pore, so vesicles detach from the mem-

brane without fusing with it [57]. Vesicle detachment

occurs with the participation of the GTPase dynamin

and similar mechanoenzymes, whose oligomers form

a constricting “collar” at the neck between the SV

and AZ membrane. In cerebrocortical terminals and

hippocampal neurons, these rapid cycles of exocytosis

and endocytosis (kiss-and-run) can sustain RRP recy-

cling for a long time [58-60].

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Ultrafast endocytosis also ensures rapid SV re-

cycling, but operates via a different mechanism. In

this case, vesicles fully fuse with the membrane, af-

ter which the membrane is retrieved through the ac-

tin-dependent formation of endocytotic invaginations

that are routed into endosomes, from which new

vesicles subsequently bud off [16, 61]. This process

is highly temperature-sensitive (Q

  ≈  3.5) and is ob-

served primarily at physiological temperatures [62].

In addition to fast endocytosis pathways, there is

a mechanism that accelerates vesicle reformation from

fragments of the presynaptic membrane. The presyn-

aptic membrane contains specialized microdomains

with pre-sorted and clustered SV proteins, referred to

as readily retrievable pool (RRetP)[63]. These regions

contain vesicular proteins, such as synaptotagmin  1,

synaptobrevin  2, and synaptophysin  1, and are also

associated with clathrin and adaptor proteins, such

as AP180 and stonins [64]. It is likely retention of ve-

sicular proteins in the limited regions of the presyn-

aptic membrane is provided by clathrin and adaptor

proteins, along with the lipid raft-dependent aggrega-

tion and interaction with cytoskeletal elements. Such

presorting ensures a high rate of clathrin-dependent

endocytosis during the initial cycles of SV exo- and en-

docytosis. Thus, the functioning of RRetP can explain

that in frog NMJs, the rate of clathrin-dependent endo-

cytosis during the first 10  s of activity is significantly

higher than its typically observed rate [63, 65].

TYPES OF NEUROTRANSMITTER

RELEASE AND THE RRP

The types of neurotransmitter release include the

following: evoked synchronous, evoked asynchronous,

and spontaneous. RRP vesicles mediate synchronous

neurotransmitter release because they are already

docked at the membrane and primed for fusion. Con-

sequently, the influx of Ca

through voltage-gated

calcium channels in the AZ can trigger their fusion

extremely rapidly (in less than 1  ms) following the

presynaptic spike. It was shown that in the hippo-

campus, synchronous vesicle fusion causes a signif-

icant decrease in the number of SVs docked to the

AZ membrane  [41]. Mutations leading to impaired

vesicle docking also reduce synchronous vesicle fu-

sion [66-68]. Additionally, evidence indicates that in

hippocampal neurons, the RRP is preferentially re-

plenished by synchronously cycling vesicles [69].

The RRP is also involved in spontaneous neu-

rotransmitter release, which may be caused by sto-

chastic fusion of SVs already in a fusion-competent

state [70]. In support of this hypothesis, studies in

the hippocampus have revealed that an increase

in the RRP size is associated with a with a higher

frequency of spontaneous neurotransmitter release

[71-74], while a decrease in the RRP size leads to

a corresponding decrease in the number of sponta-

neous release events[75]. Moreover, reduced stability

of docked SVs, such as that induced by mutations in

synaptotagmin1 (the main Ca

sensor in exocytosis),

promotes spontaneous vesicle fusion [76, 77].

However, it remains unclear whether the same

pool mediates both spontaneous and evoked release,

or there exists a distinct pool that provides most

spontaneous exocytosis events without contributing to

the evoked neurotransmitter release [78, 79]. Current

evidence supports both possibilities. One potential ex-

planation is the existence of two vesicle populations,

one of which is released constitutively regardless of

stimulation (constitutively releasing vesicles, or CRVs)

and the other is primarily responsive to stimulation,

although vesicles of this population can also fuse

spontaneously, i.e., acting as spontaneously releasing

synaptic vesicles (SRSVs)[80]. It is possible that some

RRP vesicles may belong to the SRSV pool.

The asynchronous release is mainly provided by

the recycling and reserve pools. However, in calyx

of Held synapses, some RRP vesicles undergo asyn-

chronous release during prolonged stimulation [81].

Possible molecular mechanisms of asynchronous neu-

rotransmitter release and its temporal dynamics are

described in detail in reviews [82, 83].

SYNAPTIC VESICLE POPULATIONS

UNRELATED TO THE RRP

In the first approximation, the reserve pool can

be defined as all SVs not included in the RRP. How-

ever, within this broad definition, several functional-

ly distinct subpools can be identified based on their

release properties and role in synaptic transmissions

(Fig.  2).

(I)  The recycling pool sustains synaptic trans-

mission after RRP depletion. Vesicles of the recycling

pool can be rapidly mobilized to the AZ and under-

go recycling during moderate-frequency activity, sup-

porting neurotransmission for a long time without

involving other SV populations.

(II)  The reserve pool proper supplies vesicles to

the recycling pool when the latter becomes depleted

and is mainly engaged during intense, prolonged ac-

tivity.

(III)  The spontaneously recycling pool includes

vesicles that spontaneously release neurotransmitter,

independent of the action potential.

(IV)  The resting pool contains vesicles that do

not release neurotransmitter under any condition

and likely serves as a reservoir of proteins and lipids

to meet the local needs of the presynaptic apparatus.

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Fig.  2. SV pools in the cytoplasm of the nerve terminal (excluding the RRP). The recycling pool sustains neurotransmission

during most synaptic activity regimes in vivo. However, during high-frequency stimulation, prolonged moderate activity,

or when vesicle recycling is impaired, vesicles from the reserve pool (majority of SVs in the terminal) are recruited to

support neurotransmission. Vesicles of the resting pool do not participate in recycling under normal conditions and serve

as a reservoir for proteins, lipids, and small molecules in the nerve terminal. Vesicles of the superpool are characterized

by a high mobility and can traffic between the AZs and synaptic contacts along the axon, enabling relatively rapid redis-

tribution of vesicles between distant exocytosis and endocytosis sites. Vesicles involved in evoked exocytosis, together with

a distinct population that is largely unresponsive to stimulation, may contribute to a pool that predominantly mediates

spontaneous neurotransmitter release.

(V)  The superpool (~4% of the total vesicle popu-

lation in a synapse) [5] comprises vesicles that traffic

between synaptic boutons, ensuring the optimal dis-

tribution of SVs between different, even distant, AZs.

The classification of SVs into distinct pools is

somewhat conditional. First, the same SVs can belong

to functionally different pools, i.e., mixing between

pools is possible. For example, vesicles from the re-

cycling and reserve pools can be released not only in

response to stimulation but also spontaneously, there-

by contributing to multiple functional pools. Second,

vesicles can transit between pools during synaptic ac-

tivity. During prolonged, high-frequency stimulation,

vesicles from the reserve pool are recruited into the

recycling pool. Both pools can supply vesicles to the

superpool, while reverse transition is also possible.

In addition, vesicle precursors are continuously de-

livered to the nerve terminal, where they mature

into functional SVs after several rounds of exocytosis

and endocytosis. At the same time, older vesicles are

transported back to the soma for degradation. This

ongoing turnover ensures continuous renewal and

alters the relative sizes of SV pools. Overall, intense

synaptic activity promotes mixing between vesicle

pools and facilitates adaptive changes in their rel-

ative proportions to meet the demands of synaptic

transmission.

THE RECYCLING POOL

The recycling pool includes a relatively small

number of SVs (less than 20% of the total) distributed

throughout the presynaptic terminal [2,  84].

The mechanisms of recycling pool replenishment

vary across the synapse types and activity modes. In

frog NMJs, the recycling pool sustains neurotransmis-

sion during the low-frequency stimulation (2-10  Hz)

and brief periods of high-frequency activity (tens of

seconds at 20-30  Hz), primarily through the reuse

of endocytosed vesicles. However, during prolonged

high-frequency stimulation, the recycling pool be-

comes depleted and is replaced by vesicles from the

reserve pool, which are recruited more slowly into

exocytosis, leading to a reduction in the quantal con-

tent [85,  86]. In native hippocampal synapses, the

same vesicles support neurotransmission over ex-

tended periods of time by forming a relatively closed

recycling pool maintained by endocytosis[87]. In con-

trast, in calyx of Held synapses, reserve pool vesicles

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contribute to the recycling pool replenishment even

during the low-frequency activity [45, 46].

Rapid replenishment of the RRP by the recycling

pool may depend on the interactions between SVs

and proteins intersectin  1 and endophilin. In hip-

pocampal synapses, these proteins form a dynamic

condensate that associates with SVs in the region be-

tween the RRP and reserve pool (often referred to as

the replacement zone) and promote the positioning of

vesicles in close proximity (within ~20  nm) tothe AZ.

In the absence of intersectin  1, fewer SVs are found

near the AZ, and vacant release sites are filled more

slowly, leading to the inhibition of neurotransmitter

release [88].

Vesicles of the recycling pool participate in

synchronous, asynchronous, and spontaneous neu-

rotransmitter release. In the hippocampus, the recy-

cling pool can sustain synchronous neurotransmit-

ter release over extended periods of time. However,

during high-frequency stimulation, when not all re-

lease sites can be filled with docked vesicles, neuro-

transmission is maintained through the asynchronous

release of vesicles from both the recycling and re-

serve pools [89]. Additionally, several studies indicate

that vesicles from the recycling pool also participate

in spontaneous neurotransmitter release [90, 91].

THE RESERVE POOL

Vesicles of the reserve pool are distributed

throughout the presynaptic terminal and are gener-

ally not spatially segregated from those of the recy-

cling pool [79, 92, 93]. The ratio of the reserve and

recycling pools can vary among different synaptic

boutons in the CNS [94]. Unlike the recycling pool,

reserve pool vesicles are mobilized only during pro-

longed or high-frequency stimulation, thus, suggesting

the presence of mechanisms ensuring their function-

al differentiation from the recycling pool. One such

mechanism involves association with synapsins [95],

proteins regulating vesicle mobility [96]. Interaction

with these proteins is a distinctive feature of the

reserve pool, as evidenced by studies showing that

the synapsin gene knockout leads to a marked defi-

ciency of reserve pool vesicles [97-99] and results in

rapid synaptic depression during repeated high-fre-

quency stimulation [100]. Synapsins bind reversibly

to SVs and to each other, promoting vesicle cluster-

ing. Vesicle retention may occur through two main

mechanisms: (i)  formation of tetrameric cross-links

mediated by synapsin  I, and/or (ii)  multivalent inter-

actions among synapsin molecules that drive phase

separation in the cytosol, forming a “synapsin phase”

in which SVs are embedded. The first mechanism ap-

pears to be more efficient and may dominate in ex-

citatory synapses, whereas the second one is more

likely to occur in inhibitory synapses [101].

SVs associated with synapsin are released only

when there is a sufficiently large increase in cytosolic

, which typically occurs during intense activity.

One possible explanation for why the reserve pool

is not recruited at low and medium stimulation fre-

quencies is that it includes vesicles containing pro-

teins damaged during their previous recycling, as

observed in hippocampal neurons [102].

Most reserve pool vesicles are not docked at the

membrane and, therefore, require additional time to

be transported to the AZ. This delayed recruitment

contributes to their asynchronous release in hippo-

campal synapses [103], where reduced asynchronous

release has been linked to the accumulation of ves-

icles in the reserve pool. Furthermore, reserve pool

vesicles exhibit heterogeneity in their mobilization

rates: “fast” and “slow” mobilizable vesicles replen-

ish different subpopulations of the RRP, supporting

fast and slow components of neurotransmitter re-

lease (the parallel model of vesicle delivery [104]).

Inmouse NMJs, the reserve pool contributes not only

to asynchronous release but also to synchronous exo-

cytosis [99, 105].

Vesicles of the reserve pool may vary slightly

in protein composition from those in the recycling

pool, indicating biochemical heterogeneity among

SVs. These differences can explain, at least partial-

ly, the existence of different (spontaneous or evoked)

release mechanism. For example, reserve pool vesi-

cles in the hippocampal neurons contain relatively

high amounts of the SNARE protein VAMP7, which

is associated with spontaneous exocytosis, whereas

VAMP2, a SNARE protein involved in evoked exo-

cytosis, is present at lower levels. Consequently, al-

though these vesicles are capable of stimulus-induced

release, their enrichment with VAMP7 “biases” them

toward spontaneous neurotransmitter release [91,

106]. Interestingly, vesicles generated after asynchro-

nous release induced by high-frequency stimulation

are more prone to Ca

-sensitive spontaneous release,

because both processes involve the same SNARE pro-

tein, VAMP4 [107].

The reserve pool is replenished through bulk

endocytosis (also known as activity-dependent bulk

endocytosis, ADBE), as demonstrated in many synap-

tic models, including CNS neurons [52], calyx of Held

synapses [84], and Drosophila and frog NMJs [108].

It involves membrane invagination followed by the

pinching off of cisternae, from which SVs subsequent-

ly bud off and are delivered to the reserve pool.

It is believed that this bulk endocytosis is necessary

to preserve the presynaptic membrane structure

during periods of intense stimulation of exocytosis by

maintaining its biochemical composition and tension.

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However, this pathway can also permit the entry of

high-molecular-weight structures, such as toxins and

viruses, into nerve terminals [109, 110].

Bulk endocytosis is activated in the same way

as mobilization of reserve pool SVs during high-fre-

quency stimulation  [111]. Evidence suggests that

these processes share regulatory mechanisms  [112]:

during prolonged or intense stimulation, Ca

levels

in the neuron terminal rise, leading to the activation

of calmodulin and, hence, calcineurin (protein phos-

phatase  2B) stimulation. Calcineurin dephosphorylates

synapsin, followed by its rephosphorylation by MAP

kinase and/or Cdk5, resulting in the modulation of

the reserve pool vesicle release. Concurrently, calci-

neurin dephosphorylates dynamin  1, a key step in

initiating bulk endocytosis. However, bulk endocytosis

is not the sole pathway for replenishing the reserve

pool. In calyx of Held synapses, all endocytosed vesi-

cles replenish the reserve pool, regardless of the type

of endocytosis [46].

THE SUPERPOOL AND THE RESTING POOL

The superpool is partially “extrasynaptic,” as

it represents a population of vesicles transported

along the axon between neighboring synaptic ter-

minals in an actin-dependent manner [114]. The su-

perpool is formed from vesicles of the reserve and

recycling pools, which retain their ability to release

neurotransmitters after relocation to other areas [5].

The mobility of SVs is limited due to their binding

to synapsin: disruption of synapsin functional activi-

ty leads to a massive diffusion of SVs from synaptic

boutons into axons, significantly increasing the size

of the superpool [115]. Another regulator of SV mo-

bility is the vesicular glutamate transporter (VGLUT1),

whose main function is to load neurotransmitter mol-

ecules into SVs. VGLUT1 also promotes retention of

SVs within the presynaptic terminal, but the under-

lying mechanisms remain obscure. Presumably, in-

creased SV loading, mediated by VGLUT1, limits their

mobility. Alternatively, VGLUT1 may retain SVs in

the synapse through protein–protein interactions, for

example with endophilin [116]. The superpool likely

serves as a reservoir for redistributing neurotrans-

mitter-containing vesicles between active and inactive

synapses, thereby contributing to synaptic plasticity.

An increase in the number of SVs in active synaps-

es is associated with potentiation, whereas depletion

at inactive synapses contributes to synaptic depres-

sion [114].

Some synapses contain a distinct pool of vesicles

that remains non-releasable under any conditions.

Such pool constitutes a substantial fraction of vesicles

contained in presynaptic terminals. In calyx of Held

synapses, these vesicles account for ~20% of the to-

tal pool [117], whereas in hippocampal neurons, this

proportion may reach up to 90%, according to some

estimates [118]. The functional role of this pool re-

mains debated. Studies in mouse NMJs indicate that it

may serve as a reservoir of proteins required for SV

recycling. During periods of intense stimulation, ves-

icles from this pool are not released; instead, associ-

ated soluble proteins are mobilized[119]. However, it

is still unclear whether the resting pool can be fully

mobilized. Other studies have reported that in both

calyx of Held synapses and the hippocampus, com-

plete depletion of all vesicles can occur even during

prolonged low-frequency stimulation [4, 120].

SPONTANEOUS NEUROTRANSMITTER RELEASE

AND SYNAPTIC VESICLE POOLS

Several studies suggest that spontaneous neu-

rotransmitter release is mediated by a distinct pool of

spontaneously recycling vesicles. For example, exper-

iments in inhibitory hippocampal neurons identified

two vesicular pools: one released in response to stim-

ulation, and the other released predominantly sponta-

neously [3]. Conversely, other studies indicate that a

single vesicle pool may provide both spontaneous and

evoked neurotransmitter release[121]. However, even

in the case of a single pool, the mechanisms regulat-

ing spontaneous and evoked neurotransmitter release

differ. For example, removal of membrane cholesterol

has been shown to enhance spontaneous exocytosis

while suppressing evoked neurotransmitter release in

both central synapses and NMJs [122-126].

Interestingly, in glutamatergic hippocampal syn-

apses, spontaneous neurotransmitter release can ac-

tivate receptors on the postsynaptic membrane that

remain unresponsive during evoked release  [127].

Inhibitory synapses exhibit two distinct receptor pop-

ulations: one of them responds to both evoked and

spontaneous release, while other is activated exclu-

sively by the evoked neurotransmitter release. This

segregation of receptor activation may reflect the role

of spontaneous neurotransmitter release in synapse

maturation and sustaining communication in mature

synapses [128].

Spontaneous release can occur through various

mechanisms and can be categorized into the follow-

ing types:

I.  Ca

-independent spontaneous exocytosis that

involves random fusion of docked vesicles. In this

scenario, RRP vesicles released in response to the

stimulation fuse spontaneously via random interac-

tions upon overcoming the energy barrier [70]. Be-

cause this process involves RRP vesicles, the molecu-

lar machinery mediating it is largely the same as that

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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 4 2026

of the evoked release. Recent studies in hippocampal

neurons showed that the frequency of spontaneous

exocytosis is influenced by the structure and spatial

distribution of vesicles in the nerve terminal. Within

the framework of this concept, the reserve pool im-

poses geometric constraints on the volume occupied

by more mobile recycling pool vesicles. As these vesi-

cles attempt to occupy a larger volume, they generate

an entropic force that exerts pressure on RRP vesi-

cles, thereby enhancing spontaneous exocytosis[129].

II.  Ca

-dependent spontaneous exocytosis can re-

sult from (i)  random opening of Ca

channels in the

plasma membrane[70] (for example, selective blockers

of voltage-gated Ca

channels suppress spontaneous

exocytosis in NMJs[130,  131]) or (ii)  Ca

release from

intracellular stores[132]. Stochastic fluctuations in in-

tracellular Ca

levels can trigger the fusion of docked

and undocked vesicles. It has been shown that sponta-

neous and evoked releases are triggered by different

sensors. In particular, synaptotagmin  1 and 2,

located on SVs, contribute to the synchronous release.

Doc2, located in the cytoplasm, and synaptotagmin  7,

located on the presynaptic membrane, are more sen-

sitive to Ca

concentrations and trigger spontaneous

exocytosis upon small fluctuations in the Ca

levels.

Moreover, the influx of Ca

through

different calcium

channels initiates different types of neurotransmitter

release. Voltage-gated Ca

channels primarily trigger

spontaneous release, while TRPV1 channels contrib-

ute to the asynchronous release [133]. In NMJs, re-

active oxygen species (ROS)-dependent activation of

TRPV1 channels can increase either asynchronous or

spontaneous release, depending on the primary ROS

source (mitochondria or NADPH oxidase) [124, 134].

L-type voltage-gated Ca

channels can enhance both

spontaneous and asynchronous release, as observed

in frog NMJs [135]. These findings suggest that exo-

cytosis sites may be spatially organized and more

adapted to different modes of neurotransmitter re-

lease. Such sites likely vary in their composition of

calcium channels and signaling proteins, as well as

their proximity to the endoplasmic reticulum and mi-

tochondria.

A number of studies show that some vesicles are

more prone to spontaneous fusion than others. This

difference can be attributed to the different molecu-

lar machinery involved in spontaneous versus evoked

release. For example, SNARE proteins VAMP7, VAMP4,

and vti1a are primarily responsible for spontaneous

release, whereas synaptobrevin  1 and 2 mediate

evoked release [33, 107].

These proteins are distributed across vesicles in

varying proportions. Vesicles enriched in proteins

linked to spontaneous release are more likely to un-

dergo spontaneous fusion and are thus identified

as part of the spontaneous pool. Vesicles containing

a mixture of proteins for both release types can con-

tribute to both spontaneous and evoked release with

a comparable probability.

ALTERATIONS IN THE STRUCTURE OF VESICULE

POOLS IN NEUROLOGICAL DISORDERS

Parkinson’s disease. Changes in the structure

of SV pools are among the earliest manifestations of

Parkinson’s disease, occurring even before the forma-

tion of α-synuclein aggregates that ultimately cause

the death of dopaminergic neurons. Overexpression

of α-synuclein leads to a significant increase in the

reserve pool, promoting the clumping of SVs [10, 11].

This overexpression also disrupts the mobilization of

SVs from the reserve pool to the RRP, as well as their

priming and endocytosis. The recycling pool dimin-

ishes, the number of fusion-ready vesicles decreases,

and SVs are redistributed further from the AZ. Col-

lectively, these alterations weaken neurotransmission

[136, 137].

Injection of an antibody targeting the N-terminal

domain of α-synuclein into lamprey reticulospinal syn-

apses reduced the number of SVs in both the reserve

pool and those docked at the AZ and caused dispersion

of large SV clusters into smaller ones[138]. Deletion of

α-, β-, and γ-synucleins suppressed SV inter-linking by

short connectors (as observed by cryo-electron tomog-

raphy), although the number of AZ-attached vesicles

increased [139]. Parkinson’s disease-associated muta-

tions in the α-synuclein gene inhibited endocytosis

and impaired replenishment of the RRP, as was shown

in calyx of Held terminals[140]. Likewise, α-synuclein

deletion reduced the reserve pool size and its contri-

bution to the RRP replenishment, suppressing neuro-

transmission during prolonged hippocampal synaptic

activity [141]. Furthermore, downregulation of genes

encoding Parkinson’s disease-related proteins LRRK2

(leucine-rich repeat kinase  2) and PINK1 impaired SV

mobilization from the recycling and reserve pools,

respectively [142, 143].

Therefore, alterations in the levels of α-synuclein

or its mutations disrupt the normal organization of the

reserve pool and its ability to restore the RRP during

synaptic activity. As a result, evoked neurotransmitter

release is significantly reduced even before the onset

of neuronal death.

Alzheimer’s disease. The prefibrillar amyloid

variant Aβ

1-42

, which forms at the earliest stages of

Alzheimer’s disease, impairs synaptic function in part

by disrupting vesicle transport between synaptic bou-

tons, thereby limiting synaptic plasticity. The mech-

anism is associated with the increased Ca

release

from mitochondria, which triggers hyperphosphoryla-

tion of synapsin 1 and calcium/calmodulin-dependent

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kinase  IV (CAMKIV). This disrupts the SV transport

between synapses, leading to the increased heteroge-

neity in the SV pool sizes across individual synaps-

es [9]. Pharmacological activation of CYP46A1, a key

enzyme controlling brain cholesterol turnover, re-

duced the phosphorylation of synaptic proteins and

normalized the structure of SV pools in mice over-

producing amyloid  β peptide [144, 145].

A mutation in the gene encoding BIN1, a mem-

ber of the BAR domain protein superfamily, has been

linked to an increased risk of developing late-onset

Alzheimer’s disease. In BIN1 knockout mice, excitatory

hippocampal synapses exhibit an increased number

of docked vesicles, along with the expanded reserve

pool, i.e., changes associated with the suppression of

evoked exocytosis. A decrease in the activity-depen-

dent neurotransmitter release and SV accumulation

may contribute to early memory impairment [146].

An expansion of SV pools can lead to the in-

crease in the frequency of spontaneous neurotrans-

mitter release. It has been shown that amyloid β

peptide promotes spontaneous exocytosis by increas-

ing the number of SVs and release sites [129]. At the

early disease stage, amyloid  β peptide may upregulate

the fraction of functionally active vesicles; however,

concurrent disruption of the endocytotic recycling

mechanism ultimately leads to synaptic deficit [147].

Amyloid  β oligomers were found to cause an increase

in the reserve pool size, while disrupting endocytosis

and formation of fusion-competent SVs in cultured

hippocampal neurons [148]. Over time (i.e., at the

late disease stage), these alterations should lead to

the depletion of the SV pool. Indeed, the density of

SVs in cortical axons of patients with clinically diag-

nosed Alzheimer’s disease was significantly reduced,

mainly due to a diminished reserve pool [149].

Notably, at low physiological concentrations,

amyloid β peptide (in particular, its isoforms Aβ

1-42

and Aβ

1-16

) increases the size of the recycling pool

through a mechanism associated with the stimula-

tion of cholinergic signaling, activation of α7-nic-

otinic cholinergic receptors, and subsequent cal-

cium/calcineurin-dependent dephosphorylation of

synapsin  1 [150]. Consistent with this observation,

early stages of Alzheimer’s disease are characterized

by an amyloid β-associated increase in the levels of

SV-related proteins, expansion of the total SV pool,

and presynaptic potentiation[151]. It should be noted

that enlargement of the SV pool in the absence of

impaired evoked exocytosis may enhance the learn-

ing capacity. The cohesin complex protein stromalin,

previously identified as a memory suppressor, affects

the anterograde transport of vesicles along the axon.

Dopaminergic neurons of stromalin-knockout Dro-

sophila flies exhibited an increase in the number of

vesicles in the presynaptic terminals without changes

in the number of synapses, axons, or neurons them-

selves. At the same time, mutant Drosophila flies

demonstrated an improvement in the learning abil-

ity [152].

Hence, the physiological effects of low amyloid

peptide concentrations may be associated with an

increase in the sizes of the recycling/reserve pools.

However, elevated amyloid peptide levels and asso-

ciated pathological conditions disrupt SV recycling

and promote accumulation of defective vesicles and

excessive spontaneous exocytosis, which together dis-

rupt communication between neurons.

Amyotrophic lateral sclerosis. Hyperexcitability

of motor neurons contributes to their death due to ex-

citotoxicity. An increase in the size of RRP in the spi-

nal cord excitatory synapses and spontaneous release

in NMJs were detected at the presymptomatic stage

in hSOD1(G93A) mice [13, 14]. At the same time, both

the density of vesicles in the RRP and the size of AZs

in the inhibitory nerve terminals on motor neurons

of these animals were reduced at the pre- and early

symptomatic stages. Inhibition of NO synthase mitigat-

ed these alterations in inhibitory synapses [153]. As

the disease progressed to the symptomatic stage, NMJs

exhibited further dysfunction characterized by the in-

creased NO production, suppression of synaptic ves-

icle mobilization, and diminished SV pool [154, 155].

ALS can be caused by mutations disrupting the

nuclear localization of the RNA-binding protein FUS.

These mutations induce FUS relocation to vesicles of

the reserve pool[156]. In mutant mice, synapsin  1 ex-

pression is upregulated, and synaptic vesicle recruit-

ment to exocytosis during periods of intense activity

at NMJs is suppressed [157].

Therefore, hyperactivation of motor neurons due

to a decrease in the RRP in inhibitory synapses, along-

side an increase in the RRP in excitatory synapses of

the spinal cord and suppression of SV mobilization

in NMJs, may contribute to the progression of ALS.

Schizophrenia is a polyetiological disease with

multiple contributing factors. Among these is a mu-

tation in a gene coding for the dystrobrevin-binding

protein dysbindin, which is associated with a reduced

RRP size and disruption in its replenishment during

high-frequency activity. In dysbindin-knockout mice,

these alterations cause deficits in synaptic transmis-

sion, which, in turn, can lead to various mental dis-

orders [12, 158, 159].

Memory disorders. In Drosophila melanogaster,

a single session of aversive olfactory training pro-

duces two distinct forms of memory: labile memory,

which decays within a few hours and is easily dis-

rupted after shock exposure, and stable (consolidated)

memory, which persists long term and resists to am-

nestic interference. These memory types are support-

ed by functionally distinct SV pools in the presynaptic

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terminal, and perturbations in their dynamics can

lead to memory impairment. Labile memory is as-

sociated with changes in the reserve pool. Exposure

to stressors (cold or mechanical shock) disrupt this

pool due by causing synapsin dispersion, which ulti-

mately leads to retrograde amnesia. In contrast, the

formation of consolidated memory in D.  melanogaster

critically depends on the AZ protein Rab3, which reg-

ulates recruitment of SVs to the AZ and their priming.

Rab3 modulates the size of the RRP and the prob-

ability of vesicle release – the key determinants of

synaptic plasticity [160].

Noonan syndrome belongs to the group of Ra-

sopathies, which are characterized by disruptions in

the Ras–MAPK signaling cascade. This disease is asso-

ciated with mutations in the gene of protein tyrosine

phosphatase PTPN11 and is manifested by the devel-

opmental delay and intellectual disability. Mutations

in the PTPN11 gene lead to presynaptic alterations,

including a decrease in the number of RRP vesicles

and in the total number of vesicles. In addition, both

the probability of exocytosis and the rate of synaptic

vesicle endocytosis are diminished [161].

Mitochondrial dysfunction. Mutations in the

gene of the dynamin-related protein  1 (DRP1), a key

mediator of mitochondrial division, lead to neuro-

degenerative changes. In addition to disruptions in

mitochondrial remodeling, selective deletion of the

DRP1-encoding gene in the presynaptic terminals of

calyx of Held synapses caused changes in the SV

clustering, as well as a decrease in the size of the

RRP, its replenishment, and recycling, which were ac-

companied by a reduction in the presynaptic termi-

nal volume [162]. Disruption of mitochondrial fusion

by knocking down the neuronal protein mitofusin  2

also reduced the levels of SV proteins and impaired

recruitment of SVs from the reserve pool to the RRP

during prolonged stimulation in hippocampal ax-

ons [163].

EFFECTS OF BIOLOGICALLY ACTIVE

SUBSTANCES ON SYNAPTIC VESICLE POOLS

Cannabinoids. In addition to their inhibitory ac-

tion on Ca

channels in the AZ through activation of

CB1 receptors and reduction of neurotransmitter re-

lease, cannabinoids modulate long-term plasticity by

affecting the size of vesicle pools. Activation of the G

protein-coupled CB1 receptor decreases cAMP levels

and protein kinase  A (PKA) activity. Suppression of

the PKA-mediated phosphorylation of synapsin  1 en-

hances its binding to synaptic vesicles, thus shrink-

ing the superpool. As a result, spontaneous exocytosis

decreases while the reserve pool expands, providing

a larger reservoir of vesicles available for release

during subsequent high-frequency stimulation [164].

Activation of CB1 receptors leads to the redistribu-

tion of SVs in presynaptic boutons: vesicle clustering

increases and the number of vesicles in the AZ de-

creases, indicating a transition of RRP vesicles into

the reserve pool. These changes, which depend on the

actomyosin cytoskeleton and Rho-associated kinase

signaling, lead to reduced activity at the corticostri-

atal synapses [165]. Activation of presynaptic CB1

receptors in excitatory synapses in motor neurons

decreases the RRP size, reducing synaptic transmis-

sion [166].

Reactive oxygen species enhance spontaneous

release of vesicles, which otherwise are released in

response to stimulation under normal conditions.

This phenomenon has been documented in neuro-

degenerative diseases [167]. Optogenetically induced

ROS production by presynaptic mitochondria in Dro-

sophila NMJs increased both spontaneous release and

the number of docking sites for RRP vesicles [168].

However, the application of exogenous H

and

pro-oxidants suppressed evoked neurotransmitter re-

lease and inhibited mobilization of synaptic vesicles

in NMJs [169], while endogenous stimulation of ROS

production had the opposite effect, promoting vesicle

mobilization from the reserve pool [170-173].

Zinc ions are concentrated in a subset of ZnT3

transporter-expressing SVs in glutamatergic and GAB-

Aergic synapses [174, 175]. These vesicles undergo

recycling with the involvement of the adaptor pro-

tein AP-3 [176, 177], which mediates SV formation

from endosomes and is primarily associated with

asynchronous neurotransmitter release [178]. A sub-

population of vesicles containing Zn

releases neu-

rotransmitters predominantly during high-frequency

stimulation [175]. At low nanomolar concentrations,

suppresses the mobilization of vesicles from the

reserve pool during high-frequency stimulation and

reduces the size of the RRP, which correlates with

a decrease in evoked synchronous exocytosis and

spontaneous exocytosis in NMJs [179]. Hypothetical-

ly, low Zn

concentrations can act as regulators of

the RRP size and SV mobilization through a negative

feedback mechanism, thus limiting neurotransmis-

sion. Although the presence of ZnT3 in SVs in mo-

tor nerve terminals has not been reported so far, the

main source of free Zn

may be active muscle fibers.

At high micromolar concentrations, Zn

enhanc-

es spontaneous neurotransmitter release in NMJs and

central synapses [180, 181]. This effect may result

from upregulated SV docking in the AZ due to the

increased ability of vesicular synaptotagmin  1 to bind

to anionic lipids of the AZ membrane in the presence

of Zn

 [181]. Interestingly, Zn

protects central and

peripheral synapses from the toxic effects of Cd

one of the most common pollutants [182, 183].

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Atorvastatin, an inhibitor of cholesterol biosyn-

thesis, suppresses neurotransmitter release during pe-

riods of intense activity by reducing SV mobilization

from the reserve pool, which is associated with the

upregulation of synapsin  1 in motor nerve terminals

and decrease in the population of SVs participating

in exo- and endocytosis [8]. One possible explanation

is that retention of a greater number of SVs in the

“inactive” pool helps preserve cholesterol reserves in

vesicle membranes during cholesterol-lowering ther-

apy. At the same time, NMJs exhibit an increase in

the activity-dependent uptake of low-density lipopro-

teins (cholesterol carriers), suggesting activation of

bulk endocytosis [184]. Notably, oxidized cholesterol

derivatives at submicromolar concentrations can bidi-

rectionally regulate the size of the SV pool engaged in

exo- and endocytosis in NMJs [185-187]. One potential

mechanism of their action is modulation of biophys-

ical properties of synaptic membranes.

Fluoxetine, a selective serotonin reuptake inhib-

itor that increases serotonin levels in the synaptic

cleft, also modulates the distribution of SVs between

the vesicle pools. During high-frequency stimulation,

fluoxetine promotes the mobilization of vesicles from

the reserve pool to the recycling pool, leading to

the increase in the probability of evoked exocytosis.

Hence, the antidepressant effects of fluoxetine may

also be associated with the enhanced vesicle cycling.

Additionally, fluoxetine has been shown to increase

spontaneous neurotransmitter release, accompanied

by activation of serotonin receptors [7].

Ketone bodies. The ketogenic diet– a low-carbo-

hydrate, high-fat regimen that induces ketosis through

hepatic production of ketone bodies (acetoacetate,

β-hydroxybutyrate) – is an established treatment

for drug-resistant childhood epilepsy. Ketone bodies

modulate gene expression via epigenetic mechanisms,

including β-hydroxybutyrylation and acetylation of

histones. This leads to changes in the expression of

synaptic genes in the hippocampus and remodeling of

hippocampal synapses. The ketogenic diet reduces the

size of the RRP, resulting in a decreased glutamate

release during high-frequency stimulation, which

suppresses synaptic potentiation and shifts the short-

term plasticity toward depression. Collectively, these

changes reduce hippocampal neuronal excitability,

which is thought to underlie the antiepileptic effect

of ketogenic diet [188].

ORGANIZATION OF SYNAPTIC VESICLE POOLS

IN INDIVIDUAL SYNAPSES

Ribbon synapses are predominantly found in

structures involved in the analysis of visual and

auditory information, specifically, in auditory inner

and outer hair cells, bipolar cells, photoreceptors,

and pinealocytes. They are characterized by a spe-

cial organization of vesicle pools that facilitates rap-

id and efficient transmission of visual and auditory

signals [189, 190].

A defining feature of ribbon synapses is the pres-

ence in the AZ of synaptic ribbons, proteinaceous or-

ganelle formed mainly from the Ribeye protein, that

tether the vesicles. Based on location, these vesicles

classified as membrane-proximal, and ribbon-associ-

ated (RA) or membrane-distal. In addition, a popula-

tion of undocked vesicles is freely distributed in the

cytoplasm. Membrane-proximal vesicles reside at the

base of synaptic ribbons; they are docked to the pre-

synaptic membrane and represent functional analogs

of RRP vesicles [191]. A high density of voltage-gat-

ed calcium channels is observed near the synaptic

ribbons, facilitating rapid and synchronous fusion of

these vesicles [192].

RA vesicles are positioned slightly farther from

the plasma membrane yet remain tethered to syn-

aptic ribbons. When the pool of membrane-proximal

vesicles becomes depleted, RA vesicles rapidly replen-

ish it. The presence of synaptic ribbons allows these

vesicles to quickly form a fusion-ready SNARE com-

plex, ensuring the passage through all ATP-dependent

steps more efficiently, thus shortening the time for

RRP replenishment [191, 193].

Vesicles dispersed in the cytoplasm represent an

analog of the reserve pool [194]. However, unlike the

classical reserve pool, these vesicles are not associat-

ed with synapsin, which increases their mobility and

allows to quickly replenish the pool of vesicles asso-

ciated with synaptic ribbons [195].

Together, these three vesicle pools support se-

quential phases of neurotransmitter release at rib-

bon synapses. Membrane-proximal vesicles mediate

the fast (phasic) component of release; RA vesicles

sustain tonic neurotransmitter release during contin-

uous stimulation; and cytoplasmic vesicles maintain

neurotransmission during prolonged stimulation, con-

tributing to the third, slower component [195].

Vesicle exocytosis can occur at AZs either associ-

ated or not associated with synaptic ribbons. In the

latter case, cytoplasmic vesicles provide neurotrans-

mitter release triggered by the calcium efflux from

the endoplasmic reticulum [196]. Interestingly, this

mechanism has been found only in rods, where re-

lease from AZs not associated with synaptic ribbons

maintains basal glutamate levels in the synaptic

cleft [194].

Synaptic ribbons facilitate rapid vesicles cy-

cling [190]. During stimulation, mobile vesicles are

recruited and docked to synaptic ribbons, enabling

continuous and complete replacement of released

RRP vesicles. The presence of the RRP allows vesicles

SYNAPTIC VESICLE POOLS 573

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 4 2026

to be released synchronously long after the onset of

stimulation [191].

Spontaneous neurotransmitter release in ribbon

synapses can be either Ca

-dependent or Ca

-inde-

pendent [197]. In rods, both mechanisms are present,

whereas in cones, neurotransmitter release is pre-

dominantly Ca

-independent [194]. Ca

-dependent

vesicle fusion occurs mainly at AZs associated with

synaptic ribbons, likely due to the high density of

voltage-gated calcium channels in these regions. In

rods, by contrast, Ca

-independent spontaneous re-

lease has been observed at AZs not associated with

synaptic ribbons [197].

Another distinctive feature of ribbon synapses

is that the transmitted information is encoded not

only by the stimulus frequency but also by its am-

plitude. In other words, stronger stimuli lead to the

release of greater amounts of neurotransmitter, re-

sulting in larger amplitudes of postsynaptic respons-

es [198]. Multivesicular release, which can vary in

amplitude depending on how many vesicles are re-

leased at the same time, enables the transmission of

an ultra-rapidly changing signal to the postsynapse

within milliseconds. In contrast, frequency-based en-

coding through the exocytosis of single vesicles op-

erates on a slower timescale (seconds), strongly de-

laying afferent signals. Consequently, this mode of

release is better suited for encoding sustained, stable

stimuli [199].

Central small (hippocampal) synapses. Hippo-

campal synaptic boutons are small and contain ~100-

400 vesicles, yet they provide synaptic transmission

at a frequency of 10-100  Hz [200, 201]. Maintaining

such high-frequency activity requires efficient orga-

nization and recycling of SVs.

Hippocampal synapses contain the RRP, the su-

perpool, and the recycling, reserve, and resting pools.

RRP vesicles are docked to the plasma membrane.

Upon stimulation, these vesicles undergo cycles of

exo- and endocytosis and become part of the recy-

cling pool, which sustains synaptic activity for long

periods of time[87]. RRP vesicles in the hippocampus

are replenished through clathrin-mediated endocyto-

sis, kiss-and-run mechanism, and ultrafast endocyto-

sis [16, 49, 50]. Although RRP vesicles in the hippo-

campus are predominantly released in a synchronous

manner [41, 66-68], they also contribute to sponta-

neous neurotransmitter release [71-74].

Reserve pool vesicles constitute a substantial

fraction of the total SV population. During prolonged

neuronal activity, they sustain neurotransmission by

undergoing exocytosis. However, vesicle release from

the reserve pool is predominantly asynchronous, as

these vesicles require additional time for mobiliza-

tion, docking, and fusion [89]. The reserve pool in

the hippocampal synapses can be further divided into

two distinct subpopulations: fast- and slow-mobilizing

vesicles. These subpools participate in exocytotic and

endocytotic cycles in parallel and remain function-

ally separate, even under high-frequency stimula-

tion [104]. The reserve pool is replenished primarily

through bulk endocytosis[52]. A considerable fraction

of synaptic vesicles in hippocampal synapses remains

inactive in  vivo, forming the resting pool [118]. These

vesicles may play a role in modulating spontaneous

neurotransmitter release [129].

The relative proportions of the RRP and reserve

pool vary across different synaptic boutons in the

hippocampus. Changes in the size of vesicle pools

play an important role in the formation of synaptic

plasticity. The redistribution of vesicles between bou-

tons is provided by the superpool [5].

Calyx of Held synapses are giant glutamatergic

synapses in the auditory brainstem. The total num-

ber of vesicles in the presynaptic terminal reaches

200,000-300,000 [117], and the number of AZs is 300-

700 [202].

The RRP in this synapse is heterogeneous and

can be divided into fast- and slow-releasing com-

ponents based on the probability of exocytosis (Pr).

Vesicles with high Pr provide synchronous release in

response to a single action potential, while vesicles

with low Pr are not released during low-frequency

activity and ensure asynchronous neurotransmitter

release after tetanic stimulation [203]. The heteroge-

neity of RRP vesicle exocytosis is largely attributed

to differences in the sensitivity to cytosolic Ca

 [24].

Some studies confirm the intrinsic heterogeneity in

sensitivity among vesicles themselves [25], while

others emphasize positional heterogeneity of vesicles

located at different distances from calcium channels

[204].

AZs in calyx of Held synapses exhibit substantial

variability in the number of voltage-gated calcium

channels, ranging from 5 to 200 per AZ. This vari-

ability likely contributes to differences in local Ca

dynamics and, consequently, to diverse Pr values ob-

served among RRP vesicles [31].

Most vesicles (~80%) in the calyx of Held synaps-

es participate in the cycles of exocytosis and endocy-

tosis [4]. The recycling pool is replenished primarily

through mobilization of vesicles from the reserve

pool [44, 46], while newly endocytosed vesicles are

preferentially routed to the reserve pool [46]. During

moderate-frequency stimulation, vesicle retrieval is

predominantly mediated by clathrin-dependent en-

docytosis. At higher stimulation frequencies, faster

mechanisms, such as kiss-and-run and bulk endocy-

tosis, become more prominent [84]. The resting pool

comprises roughly 20% of the total vesicle population

and is not released even under high-frequency stim-

ulation [117].

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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 4 2026

Drosophila neuromuscular junction contains

~84,000 vesicles. The RRP constitutes 14-19% of the

total vesicle pool [205]. A distinctive feature of the

spatial organization of vesicle pools in Drosophila

NMJ is that the RRP is positioned at the periphery

of the nerve terminal, whereas the reserve pool oc-

cupies its central region [206]. During low-frequency

stimulation, RRP vesicles undergo continuous recy-

cling to sustain basal activity levels. In contrast, the

reserve pool is recruited into exocytosis and endo-

cytosis cycling primarily during periods of high-fre-

quency activity [206, 207]. Replenishment of SV pools

is mediated by several clathrin- and dynamin-depen-

dent endocytosis mechanisms [208].

The homeostatic plasticity at Drosophila NMJs is

manifested as a compensatory increase in neurotrans-

mitter release in response to the reduced postsynap-

tic receptor activity. A key mechanism underlying

presynaptic homeostatic plasticity is an expansion of

the RRP [209, 210].

Vesicles involved in evoked exocytosis are also

involved in spontaneous exocytosis. However, Dro-

sophila NMJ may contain a subset of vesicles spe-

cialized for spontaneous neurotransmission [211].

Although the same AZs can support both evoked and

spontaneous release, functional heterogeneity exists,

as some AZs preferentially mediate evoked exocyto-

sis, whereas others are more strongly associated with

spontaneous neurotransmitter release [212, 213].

Frog neuromuscular junction. Frog NMJ con-

tains hundreds of thousands of vesicles [2]. Approxi-

mately 20% of these vesicles belong to the RRP, while

the remaining form the reserve pool [20]. In frog

NMJs, the RRP and reserve pools operate in parallel

way without overlapping [85], and distinct exocytosis

sites likely mediate release from each pool.

The RRP is distributed throughout the nerve ter-

minal [20]. During low-frequency activity, RRP ves-

icles can be recycled for extended periods of time

without involvement of the reserve pool, as they are

replenished via endocytosis. Upon high-frequency

stimulation (30  Hz), the RRP becomes depleted after

10-20  s, but is fully recovered within 1 min through

endocytosis. Reserve pool vesicles do not contribute

to the RRP replenishment [85].

The reserve pool participates in synaptic trans-

mission during high-frequency stimulation (20-30  Hz)

and undergoes recycling in parallel with the RRP, as

vesicles retrieved by endocytosis are returned to the

reserve pool[85]. The reserve pool is replenished pri-

marily through bulk endocytosis [108]. After endocy-

tosis, vesicles originating from the two pools remain

segregated and do not mix [17].

At the NMJ, the same vesicles participate in both

synchronous and asynchronous exocytosis [214]. Dif-

ferent subtypes of Ca

channels may be preferential-

ly involved in these two forms of release [134, 135].

Spontaneous release of vesicles is Ca

-dependent and

is driven by both intracellular Ca

[215] and exog-

enous Ca

entering through Ca

channels of the

plasma membrane (e.g., L-type channels) [130, 135].

Depolarization can also enhance spontaneous exocy-

tosis in a Ca

-independent manner [216].

Mouse neuromuscular junction. Mouse motor

nerve terminals contain approximately 400-850 AZs,

with 1-2 vesicles per each AZ. Accordingly, the RRP

comprises ~800-1700 vesicles, with the total vesicle

population in the NMJ exceeding 400,000-800,000.

Vesicle participation in exocytosis strongly depends

on the stimulation frequency. At low frequencies,

only a subset of docked RRP vesicles undergoes exo-

cytosis and recycling, whereas high-frequency activity

recruits nearly entire RRP [22, 24, 217].

Mouse NMJs exhibit pronounced frequency-de-

pendent mobilization of SVs. Thus, mouse nerve ter-

minals contain a “housekeeping” vesicle pool that

equally sustains neurotransmission upon both low-

and high-frequency stimulation. However, efficient

transmission during high-frequency activity requires

recruitment of an additional “plug-in” pool that re-

mains largely inactive at low frequencies [218]. The

contribution of this auxiliary pool is enhanced by ac-

tivation of β2-adrenergic receptors [219].

The type of endocytosis used for the pool replen-

ishment also depends on the stimulation frequency.

At moderate frequencies, exocytosis primarily oc-

curs via full fusion of vesicles with the presynap-

tic membrane. Under high-frequency conditions,

faster recycling mechanisms, such as kiss-and-run,

become more prominent [56, 220]. After high-fre-

quency stimulation, vesicle retrieval occurs through

both clathrin-dependent and clathrin-independent

endocytosis [110, 220, 221], while bulk endocytosis

is activated during intense stimulation of the motor

nerve [110, 222].

Reserve pool vesicles participate in both syn-

chronous and asynchronous neurotransmitter release

[99, 105]. Evidence suggests that largely overlapping

vesicle populations participate in both evoked and

spontaneous exocytosis [223].

CONCLUSION

Efficient synaptic transmission relies on contin-

uous recycling of vesicles from functionally different

pools. RRP vesicles are primed for fusion with the

presynaptic membrane, enabling rapid, synchronous

neurotransmitter release in response to stimulation.

The recycling pool sustains transmission during mod-

erate stimulation and short periods of high-frequen-

cy stimulation, whereas the reserve pool supports

SYNAPTIC VESICLE POOLS 575

BIOCHEMISTRY (MOSCOW) Vol. 91 No. 4 2026

prolonged neurotransmission when recycling vesi-

cle pool becomes depleted. In addition, a subset of

vesicles serves as a reservoir of lipids and proteins,

ensuring a rapid supply of essential components for

synaptic function. The superpool provides the redis-

tribution of vesicles between active and inactive syn-

aptic boutons, while the spontaneous pool contributes

to synapse maturation and maintenance of baseline

activity.

Differences in the cycling of vesicles belonging to

different pools enable synapses to adapt to varying

levels of activity. Under resting conditions and upon

moderate stimulation, vesicle replenishment occurs

primarily via clathrin-mediated endocytosis. During

high-frequency stimulation, faster mechanisms, such

as kiss-and-run and ultrafast endocytosis, allow vesi-

cle pool recovery in a short period of time. Bulk en-

docytosis preserves presynaptic membrane integrity

during periods of intense exocytosis.

The presence in presynaptic terminals of func-

tionally heterogeneous populations of vesicles, dif-

fering in their capacity for exocytosis, mobilization,

and recycling kinetics, together with the existence of

multiple modes of exo- and endocytosis, provides a

fundamental basis for synaptic plasticity and adapta-

tion of the synaptic apparatus.

The importance of these mechanisms is empha-

sized by the fact that disruptions in the vesicle cycling

and organization of SV pools contribute significantly

to the pathogenesis of numerous neurological and

neurodegenerative disorders, including Alzheimer’s

disease, Parkinson’s disease, schizophrenia, and ALS.

Moreover, SV dynamics and recycling mechanisms

represent targets for various pharmacological agents,

such as fluoxetine and atorvastatin.

Further investigation into the organization and

functional properties of SV pools is essential for a

comprehensive understanding of neuronal communi-

cation under both physiological and pathological con-

ditions. In particular, the mechanisms governing the

formation and reorganization of vesicle pools consti-

tute promising targets for therapeutic intervention.

However, the processes underlying the genesis of SV

pools remain largely unexplored.

Unresolved fundamental questions remain: which

molecular mechanisms determine the assignment of

SVs to specific pools; how the organization of these

pools is preserved despite repeated reuse of the same

vesicles; which regulatory systems govern the pool

size and mediate vesicle exchange between the pools;

and what underlies the heterogeneity within indi-

vidual pools, particularly the RRP and reserve pool.

It is also unclear how SV pools are altered under

pathological conditions associated with mitochondrial

dysfunction, endoplasmic reticulum stress, impaired

autophagy, and disruptions of the endolysosomal

system. Given that SVs are composed of substantial

amounts of proteins and lipids and undergo contin-

uous cycles of exocytosis and endocytosis, the func-

tioning of SV pools is likely tightly coupled to cellular

metabolic processes. Elucidating these relationships

could substantially advance our understanding of

the global interplay between metabolism and synap-

tic function. Moreover, identifying mechanisms that

regulate the behavior of distinct vesicle pools and

the rate of SV recycling may suggest new therapeutic

strategies for a broad range of neurological and neu-

rodegenerative disorder

Abbreviations

AZ active zone

NMJ neuromuscular junction

RRP readily releasable pool

SV synaptic vesicle

Acknowledgments

The authors are grateful to A.  N.  Tsentsevitsky (Kazan

Institute of Biochemistry and Biophysics) for valuable

comments and discussion.

Contributions

A.M.P. developed the study concept and edited the

manuscript; C.R.G. wrote and edited the text of the

article.

Funding

This publication was supported by the Brain Program

of the Idea Scientific Center for Advanced Interdisci-

plinary Research and State Assignment for the Kazan

Scientific Center.

Ethics approval and consent to participate

This work does not contain studies involving human

or animal subjects.

Conflict of interest

The authors of this work declare that they have no

conflicts of interest.

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