Synaptic Vesicle Recycling at the Developing Presynapse

“All the SNARE proteins contain motifs that facilitate a processive association (colloquially termed ‘zippering’) that drives the fusion of the SV with the plasma membrane (Rizo et al. 2024; Weber et al. 1998).”

“A key aspect of CME is the clustering of SV cargo proteins by adaptor protein complex 2 (AP2), which both identifies cargo for retrieval and the recruitment of subsequent clathrin coat proteins (Kelly and Owen 2011; McMahon and Boucrot 2011; Robinson 2004).”

“Rodents display a much earlier peak of synaptogenesis, which is typically maximal at the second to third postnatal week, although some recent publications report steady synapto-genesis until young adulthood (Semple et al. 2013; Wildenberg et al. 2023).”

“However, this result contrasts with initial studies in the Munc-18 knock-out mouse, which showed intact initial synaptogenesis despite the absence of neurotransmission (Verhage et al. 2000).”

“The recycling pool can be subdivided into the readily releasable pool (RRP), which as the name suggests, are the SVs that fuse first on action potential invasion (Rosenmund and Stevens 1996) and the reserve pool (which repopulate the RRP).”

“The nomenclature of these SV populations is not uniform, but typically they can be subdivided into SVs that are either accessible (recycling pool) or inaccessible (resting pool) to neuronal activity (Rizzoli and Betz 2005).”

“…intracellular free calcium ([Ca 2+ ] i ) (Neher and Sakaba 2008), which is sensed by the calcium trigger for synchronous neurotransmitter release, synaptotagmin-1 (Syt1) that binds calcium with extremely low affinity (Fernández-Chacón et al. 2001; Geppert et al. 1994; Nishiki and Augustine 2004).”

“The spatial organisation results in the creation of a transient microdomain of extremely high intracellular free calcium ([Ca 2+ ] i ) (Neher and Sakaba 2008), which is sensed by the calcium trigger for synchronous neurotransmitter release, synaptotagmin-1 (Syt1) that binds calcium with extremely…”

“A key aspect of CME is the clustering of SV cargo proteins by adaptor protein complex 2 (AP2), which both identifies cargo for retrieval and the recruitment of subsequent clathrin coat proteins (Kelly and Owen 2011; McMahon and Boucrot 2011; Robinson 2004).”

“CME is an established mechanism of cargo retrieval in all mammalian cells and was originally proposed to be the dominant mode of SV retrieval at the presynapse (Granseth et al. 2006).”

“This influx of calcium is spatially regulated, with a high concentration of voltage-gated calcium channels (VGCCs) anchored at the active zone, which is the site of neurotransmitter release (Gundelfinger and Fejtova 2012; Simms and Zamponi 2014).”

“The major [Ca 2+ ] i sink in nerve terminals is mitochondria, which limit action potential-evoked [Ca 2+ ] i increases in nerve terminals (Devine and Kittler 2018).”

“In all cases, regenerated SVs are uncoated, acidified and filled with neurotransmitters to replenish SV pools. allowed the visualisation of endocytic intermediates just 50 ms after stimulation (Watanabe, Liu, et al. 2013; Watanabe, Rost, et al. 2013; Watanabe et al. 2014).”

“…as depletion of available SVs) attenuates over development, suggesting that there is faster replenishment of SV pools in mature neurons (Bolshakov and Siegelbaum 1995; Cheetham and Fox 2010; Crins et al. 2011; Frick et al. 2007; Iwasaki and Takahashi 2001; Taschenberger and Von Gersdorff 2000).”

“This results in the direct generation of individual SVs from the plasma membrane by invagination and then fission of clathrin-coated SVs mediated by the GTPase dynamin-1 (Antonny et al. 2016).”

“Other key regulators are calcium pumps on both the plasma membrane and endoplasmic reticulum, which sequester [Ca 2+ ] i (Billups and Forsythe 2002; Chanaday and Kavalali 2022; Wan et al. 2012).”

“…viewed as depletion of available SVs) attenuates over development, suggesting that there is faster replenishment of SV pools in mature neurons (Bolshakov and Siegelbaum 1995; Cheetham and Fox 2010; Crins et al. 2011; Frick et al. 2007; Iwasaki and Takahashi 2001; Taschenberger and Von…”

“As stated in the introduction, various modes of endocytosis are proposed to coexist at the presynapse, namely CME, ADBE and UFE.”

“Central to this mechanism is the coordinated dephosphorylation of a subset of endocytosis proteins called the dephosphins, by the calcium-dependent protein phosphatase calcineurin (Clayton and Cousin 2009; Cousin and Robinson 2001; Imoto et al. 2022, 2024).”

“This confirmed earlier studies that monitored SV recycling using antibodies directed against the lumenal domain of Syt1 (Matteoli et al. 1992).”

“Synaptogenesis starts as early as five gestational weeks in humans and peaks during the first two postnatal years (Budday et al. 2015).”

“A leading candidate is Doc2, which is a cytoplasmic C2 domain-containing protein that has affinity for calcium at least two orders of magnitude higher than Syt1 (Groffen et al. 2010).”

“…horn retain sensitivity to ωCgTx-GVIA to maturity, as do non-NMDA ( N - methyl- d-aspartate) excitatory synapses in cerebral cortical neurons according to whole-cell recordings of evoked excitatory postsynaptic currents (EPSCs) and inhibitory postsynaptic currents (IPSCs) (Iwasaki et al. 2000).”

“As mentioned earlier, this is triggered by the binding of free calcium ions to Syt1, which drives altered interactions with both the assembled SNARE complex and plasma membrane, resulting in the synchronisation of neurotransmitter release to neuronal activity (Hui et al. 2009; Toulmé et al. 2024).”

“…P/Q-and N-type VGCCs account for the majority of the evoked calcium influx in both excitatory and inhibitory mammalian presyn-apses, which was revealed by exploiting pharmacological blockers for VGCC subtypes or by knocking out a specific VGCC subunit (Dolphin and Lee 2020; Reid et al. 2003).”

“As stated above, inhibition of voltage-gated sodium channels in immature neurons using TTx had little effect on presynaptic activity (Andreae et al. 2012; Groc et al. 2002; Hsia et al. 1998).”

“With increasing maturity, this phenomenon tapers off around the second postnatal week in rodents (Blankenship and Feller 2010; Kersbergen and Bergles 2024; Luhmann et al. 2016; Pires et al. 2021).”

“…by calcium influx, but whether the calcium influx is generated by VGCCs or other sources remains controversial, especially in glutamatergic synapses (Babiec and O'Dell 2018; Chanaday et al. 2021; Dai et al. 2015; Liu et al. 2018; Reese and Kavalali 2015; Williams and Smith 2018; Xu et al. 2009).”

“A key component of this is the delivery and tethering of VGCCs (Chin and Kaeser 2024; Held et al. 2020), with the latter requiring interactions with a series of cell adhesion molecules, such as the neurexins and laminins (Chen et al. 2011; Luo et al. 2020; Nishimune et al. 2004).”

“As stated in the introduction, various modes of endocytosis are proposed to coexist at the presynapse, namely CME, ADBE and UFE.”

“For more extensive coverage of these specific topics, refer to other reviews (Leterrier 2018; Nozumi and Igarashi 2018; Petzoldt 2023; Rizalar et al. 2021).”

“In all cases, regenerated SVs are uncoated, acidified and filled with neurotransmitters to replenish SV pools. allowed the visualisation of endocytic intermediates just 50 ms after stimulation (Watanabe, Liu, et al. 2013; Watanabe, Rost, et al. 2013; Watanabe et al. 2014).”

“The authors hypothesised that the VGCC clusters regulate the fusion of SVs within a certain distance, which was simulated in silico (Nakamura et al. 2015).”

“For example, depletion of clathrin heavy chain arrests SV generation from endosomes generated via UFE (Watanabe et al. 2014), in an identical manner to ADBE (Ivanova et al. 2021; Kononenko et al. 2014).”

“…cerebellar and thalamic synapses and excitatory neuro-transmission in the brainstem displayed decreasing sensitivity to the cone snail toxin ω- conotoxin-GVIA (ωCgTx-GVIA, which blocks N-type VGCCs) in the second postnatal week in rat brain slices (Iwasaki et al. 2000; Iwasaki and Takahashi 1998).”

“…the plasma membrane of the release site, while priming renders SVs fusion-competent by changing the conformation of the SNARE complex, a process coordinated via interactions with a series of molecules including Munc-13, Munc-18, complexin and Syt1 (An and Lindau 2024; Imig et al. 2014; Rizo 2022).”

“Although not covered in this review, the delivery and maturation of the active zone machinery are key initial steps in the establishment of the presynapse (Bolz and Haucke 2024; Gundelfinger et al. 2016).”

“In more detail, processes start to develop in culture within 2 days after plating, but axonal identity is established with the localisation of the axon initial segment starting from around 5 days in vitro (DIV) (Bolz and Haucke 2024; Dotti et al. 1988; Yang et al. 2007).”

“…P/Q-and N-type VGCCs account for the majority of the evoked calcium influx in both excitatory and inhibitory mammalian presyn-apses, which was revealed by exploiting pharmacological blockers for VGCC subtypes or by knocking out a specific VGCC subunit (Dolphin and Lee 2020; Reid et al. 2003).”

“…primary hippocampal cultures were challenged with high-frequency action potential trains in the presence of the fluid-phase marker te-tramethylrhodamine (TMR)- dextran, which is more likely to be internalised via ADBE rather than CME due to size exclusion (Clayton et al. 2008; Kim et al. 2024).”

“Manipulating SNA through genetic or pharmacological methods showed topological disruptions in various systems, such as defective segregation of the visual pathway, as reviewed previously (Martini et al. 2021; Wu, Kourdougli, and Portera-Cailliau 2024).”

“While its initiation is speculated to be driven by spontaneous SV exocytosis, its propagation is likely to be mediated by evoked SV exocytosis of neurotransmitters with an additional contribution from gap junctions (Martini et al. 2021; Opitz et al. 2002; Pires et al. 2021).”

“Similar to UFE, it has a two-step mechanism involving initial endosome formation followed by generation of individual SVs from the endosomal structures (Clayton and Cousin 2009).”

“For example, in mature nerve terminals, a series of v-SNARE proteins including Vti1a, VAMP4 and VAMP7 all appear to bias neurotransmitter release towards spontaneous SV fusion when overexpressed in primary neuronal cultures (Bal et al. 2013; Lin et al. 2020; Ramirez et al. 2012).”

“The increased coupling through maturity is not exclusive to excitatory synapses, since inhibitory neurotransmission from Basket cells to Purkinje cells in cerebellar slices also displayed reduced sensitivity to EGTA from measurements at P7–9 to those at P14–16 (Chen et al. 2024).”

“This cis-SNARE complex is disrupted by the action of the ATPase NSF, which is recruited via soluble NSF attachment proteins (Yoon and Munson 2018).”

“Intriguingly, some atypical SNARE and Syt proteins, such as VAMP4 and Syt7, may bias the triggering of different endocytosis modes in an indirect manner via their control of asynchronous release (Li et al. 2017; Lin et al. 2020; Raingo et al. 2012).”

“The calcium dependency of this control has been debated (Pang et al. 2011); however, it was later explained due to gain of function mutations within the calcium binding motif in molecular replacement studies (Courtney et al. 2018).”

“…intracellular free calcium ([Ca 2+ ] i ) (Neher and Sakaba 2008), which is sensed by the calcium trigger for synchronous neurotransmitter release, synaptotagmin-1 (Syt1) that binds calcium with extremely low affinity (Fernández-Chacón et al. 2001; Geppert et al. 1994; Nishiki and Augustine 2004).”

“There is also growing evidence that the calcium required for miniature EPSCs (mEPSCs) is derived from VGCCs (Ermolyuk et al. 2013; Lee et al. 2022).”

“Two later studies examined whether this was due to a change in the spatial topography of the active zone or an intrinsic change in the release machinery using patch clamp electrophysiology, calcium uncaging and calcium dye imaging (Kochubey et al. 2009; Wang et al. 2008).”

“Regardless of the mode of SV fusion, SV endocytosis is an essential step to regenerate SVs to replenish SV pools and maintain plasma membrane tension (Denker et al. 2011; Gan and Watanabe 2018).”

“This influx of calcium is spatially regulated, with a high concentration of voltage-gated calcium channels (VGCCs) anchored at the active zone, which is the site of neurotransmitter release (Gundelfinger and Fejtova 2012; Simms and Zamponi 2014).”

“For example, in mature nerve terminals, a series of v-SNARE proteins including Vti1a, VAMP4 and VAMP7 all appear to bias neurotransmitter release towards spontaneous SV fusion when overexpressed in primary neuronal cultures (Bal et al. 2013; Lin et al. 2020; Ramirez et al. 2012).”

“UFE and ADBE may also share the same molecular mechanism, although they were originally proposed to be triggered by mild and intense stimulation respectively (Blumrich et al. 2023; Clayton and Cousin 2009; Imoto et al. 2022, 2024; Smillie et al. 2005; Watanabe et al. 2018; Xue et al. 2011).”

“Regardless of the mode of SV fusion, SV endocytosis is an essential step to regenerate SVs to replenish SV pools and maintain plasma membrane tension (Denker et al. 2011; Gan and Watanabe 2018).”

“At the Calyx of Held, EPSC amplitude in response to axonal stimulation was decreased by EGTA before hearing onset at P8–12 but not at P16–18, when hearing begins (Fedchyshyn and Wang 2005).”

“For example, it appears to be required for the clustering of postsynaptic receptors (Saitoe et al. 2001).”

“A key aspect of CME is the clustering of SV cargo proteins by adaptor protein complex 2 (AP2), which both identifies cargo for retrieval and the recruitment of subsequent clathrin coat proteins (Kelly and Owen 2011; McMahon and Boucrot 2011; Robinson 2004).”

“UFE and ADBE may also share the same molecular mechanism, although they were originally proposed to be triggered by mild and intense stimulation respectively (Blumrich et al. 2023; Clayton and Cousin 2009; Imoto et al. 2022, 2024; Smillie et al. 2005; Watanabe et al. 2018; Xue et al. 2011).”

“It is likely that CME is a basal mechanism that enables membrane homeostasis and axon guidance from the early stage of presynaptic differentiation, as demonstrated in the growth cone (Tojima et al. 2010).”

“On the contrary, SV endocytosis at the Calyx of Held synapse displayed reduced sensitivity to calmodulin and CaN antagonists from P7–9 to P13–14, when SV retrieval was monitored using membrane capacitance as a surrogate for surface area (Yamashita et al. 2010).”

“Neurotransmitter is loaded into SVs against its concentration gradient by exploiting a pro-tonmotive force generated by a V-type ATPase located on the SV membrane (Gowrisankaran and Milosevic 2020; Pulido and Ryan 2021).”

“For example, it appears to contribute to presynaptic development in Drosophila (Cho et al. 2015; Choi et al. 2014).”

“…by calcium influx, but whether the calcium influx is generated by VGCCs or other sources remains controversial, especially in glutamatergic synapses (Babiec and O'Dell 2018; Chanaday et al. 2021; Dai et al. 2015; Liu et al. 2018; Reese and Kavalali 2015; Williams and Smith 2018; Xu et al. 2009).”

“…knock-down of its heavy or light chain has been shown to have either little impact (Kononenko et al. 2014; López-Hernández et al. 2022; Soykan et al. 2016) or retard the retrieval of various pHluorin reporters (López-Hernández et al. 2022; Lopez-Murcia et al. 2014; Nicholson-Fish et…”

“The selective elimination of evoked neurotransmitter release by either expression of tetanus toxin light chain (to cleave neuronal(n)- synaptobrevin) or membrane-tethered Plectreurys toxin II (which blocks cacoph-ony, a Drosophila N-type VGCC that regulates evoked but not spontaneous release) had no impact on bouton n…”

“…into the plasma membrane and becomes fluorescent) was dependent on calcium in 2- day-old Xenopus spinal cord neurons, and the internalisation of Syt1 antibodies was also dependent on the extracellular calcium concentration at DIV 2–4 in hippocampal neurons (Coco et al. 2002; Dai and Peng 1996).”

“Intriguingly, some atypical SNARE and Syt proteins, such as VAMP4 and Syt7, may bias the triggering of different endocytosis modes in an indirect manner via their control of asynchronous release (Li et al. 2017; Lin et al. 2020; Raingo et al. 2012).”

“The calcium dependency of this control has been debated (Pang et al. 2011); however, it was later explained due to gain of function mutations within the calcium binding motif in molecular replacement studies (Courtney et al. 2018).”

“It was suggested that the rearrangement of VGCC localisation at the active zone was the driving force behind reduced ωCgTx-GVIA sensitivity at maturing nerve terminals (Pravettoni et al. 2000).”

“As stated above, inhibition of voltage-gated sodium channels in immature neurons using TTx had little effect on presynaptic activity (Andreae et al. 2012; Groc et al. 2002; Hsia et al. 1998).”

“A key component of this is the delivery and tethering of VGCCs (Chin and Kaeser 2024; Held et al. 2020), with the latter requiring interactions with a series of cell adhesion molecules, such as the neurexins and laminins (Chen et al. 2011; Luo et al. 2020; Nishimune et al. 2004).”

“A key component of this is the delivery and tethering of VGCCs (Chin and Kaeser 2024; Held et al. 2020), with the latter requiring interactions with a series of cell adhesion molecules, such as the neurexins and laminins (Chen et al. 2011; Luo et al. 2020; Nishimune et al. 2004).”

“…as depletion of available SVs) attenuates over development, suggesting that there is faster replenishment of SV pools in mature neurons (Bolshakov and Siegelbaum 1995; Cheetham and Fox 2010; Crins et al. 2011; Frick et al. 2007; Iwasaki and Takahashi 2001; Taschenberger and Von Gersdorff 2000).”

“…its heavy or light chain has been shown to have either little impact (Kononenko et al. 2014; López-Hernández et al. 2022; Soykan et al. 2016) or retard the retrieval of various pHluorin reporters (López-Hernández et al. 2022; Lopez-Murcia et al. 2014; Nicholson-Fish et al. 2015; Wei et al. 2024).”

“First, when evoked EPSC amplitude and presynaptic calcium charge were correlated, the same amount of calcium influx resulted in increased glutamate release at P12–15 synapses compared to those from P7–10 (Kochubey et al. 2009).”

“Individual SVs are generated from the bulk endosome through similar cargo recognition mechanisms as CME (clathrin and adaptor proteins) (Cheung and Cousin 2012; Ivanova et al. 2021; Kononenko et al. 2014), but the endosome also serves as a sorting platform to recycle or degrade cargo proteins via…”

“This increased coupling occurred coincident with an increasing contribution from P/Q-type VGCCs (Bornschein et al. 2019).”

“In primary neurons from dissociated hippocampal culture, the transition in SV recycling mechanisms from spontaneous to evoked was highlighted in studies where SV turnover was revealed by biosyn labelling (Andreae et al. 2012).”

“Clues to the mode of endocytosis that is activated early in development could come from growth cones, since they display membrane turnover and appear to have discrete constitutive and evoked pathways (Cheng and Reese 1987; Diefenbach et al. 1999; Nozumi and Igarashi 2018).”

“…by calcium influx, but whether the calcium influx is generated by VGCCs or other sources remains controversial, especially in glutamatergic synapses (Babiec and O'Dell 2018; Chanaday et al. 2021; Dai et al. 2015; Liu et al. 2018; Reese and Kavalali 2015; Williams and Smith 2018; Xu et al. 2009).”

“For more extensive coverage of these specific topics, refer to other reviews (Leterrier 2018; Nozumi and Igarashi 2018; Petzoldt 2023; Rizalar et al. 2021).”

“In contrast to the previous study, the ablation of CaN A ɑ expression resulted in similar defects in SV endocytosis at both time points (Wu et al. 2014).”

“Numerous studies suggest that synaptobrevin-2 and other SV cargo proteins are clustered at the periphery of the active zone for subsequent retrieval by endocytosis (Cousin 2017; Gimber et al. 2015).”

“…as depletion of available SVs) attenuates over development, suggesting that there is faster replenishment of SV pools in mature neurons (Bolshakov and Siegelbaum 1995; Cheetham and Fox 2010; Crins et al. 2011; Frick et al. 2007; Iwasaki and Takahashi 2001; Taschenberger and Von Gersdorff 2000).”

“The authors tested membrane capacitance at the Calyx of Held at P7-10 and P13-14, which marks before and after hearing onset, concomitant with tighter calcium coupling to SV exocytosis.”

“This finding contrasts with studies in organotypic hippocampal slice cultures, which revealed a sevenfold acceleration in the rate of synaptophysin-pHluorin quenching between DIV 5–7 to DIV 14–28 (Rose et al. 2013).”

“…(mIPSCs), primarily due to studies that revealed a decrease in mIPSC frequency in cortical and hippocampal cultured neurons when recorded in the presence of VGCC blockers such as ωCgTx-GVIA and ω- Agatoxin-IVA (ωAgTx-IVA, which inhibits P/Q-type VGCCs) (Goswami et al. 2012; Williams et al. 2012).”

“These endosomes could subsequently be stimulated to fuse and release dye on further KCl challenge (Diefenbach et al. 1999).”

“…through similar cargo recognition mechanisms as CME (clathrin and adaptor proteins) (Cheung and Cousin 2012; Ivanova et al. 2021; Kononenko et al. 2014), but the endosome also serves as a sorting platform to recycle or degrade cargo proteins via the endolysosomal system (Ivanova and Cousin 2022).”

“The same experiment in layer 5 pyramidal neurons showed no significant difference in the replenishment rate of the RRP from P8–10 to P21–24 (Bornschein et al. 2020; Bornschein and Schmidt 2019).”

“More recent studies revealed the mechanism un-derpinning this basal membrane turnover, using uptake of a related dye, FM4-64, at growth cones of cultured hippocampal neurons (Bonanomi et al. 2008).”

“It is possible that the molecular machinery for ADBE is present at the presynapse earlier in development; however, it is only triggered by the sudden increase in plasma membrane surface area generated by synchronous SV exocytosis (Ogunmowo et al. 2023).”

“The same experiment in layer 5 pyramidal neurons showed no significant difference in the replenishment rate of the RRP from P8–10 to P21–24 (Bornschein et al. 2020; Bornschein and Schmidt 2019).”

“The ability to patch clamp this atypical nerve terminal facilitates delivery of small molecules directly to the presynapse and permits direct measurements of both calcium influx and SV fusion (via membrane capacitance) (Neher 2017).”

“All the SNARE proteins contain motifs that facilitate a processive association (colloquially termed ‘zippering’) that drives the fusion of the SV with the plasma membrane (Rizo et al. 2024; Weber et al. 1998).”

“…which is action potential-independent but calcium-dependent fusion of SVs, while ac-knowledging evidence for calcium-independent spontaneous SV fusion events, which are observed frequently in developing neurons (Glitsch 2008; Liu et al. 2018; Truckenbrodt and Rizzoli 2014; Williams and Smith 2018).”

“The early treatment of APV and CNQX resulted in a decrease in dendrite length and in dendritic branching, which was distinct from the TTx-treated group (Andreae and Burrone 2015).”

“Parallel studies examining the uptake of large dextran in chick dorsal root ganglion neurons supported the presence of a mac-ropinocytosis pathway, with uptake inhibited by antagonists selective for membrane ruffling (Kabayama et al. 2009, 2011).”

“Parallel studies examining the uptake of large dextran in chick dorsal root ganglion neurons supported the presence of a mac-ropinocytosis pathway, with uptake inhibited by antagonists selective for membrane ruffling (Kabayama et al. 2009, 2011).”

“Numerous studies suggest that synaptobrevin-2 and other SV cargo proteins are clustered at the periphery of the active zone for subsequent retrieval by endocytosis (Cousin 2017; Gimber et al. 2015).”

“Other key regulators are calcium pumps on both the plasma membrane and endoplasmic reticulum, which sequester [Ca 2+ ] i (Billups and Forsythe 2002; Chanaday and Kavalali 2022; Wan et al. 2012).”

“…by calcium influx, but whether the calcium influx is generated by VGCCs or other sources remains controversial, especially in glutamatergic synapses (Babiec and O'Dell 2018; Chanaday et al. 2021; Dai et al. 2015; Liu et al. 2018; Reese and Kavalali 2015; Williams and Smith 2018; Xu et al. 2009).”

“…are generated from the bulk endosome through similar cargo recognition mechanisms as CME (clathrin and adaptor proteins) (Cheung and Cousin 2012; Ivanova et al. 2021; Kononenko et al. 2014), but the endosome also serves as a sorting platform to recycle or degrade cargo proteins via the…”

“The Calyx of Held is an atypical synapse because of its giant size and its high number of active zones (Baydyuk et al. 2016).”

“Other key regulators are calcium pumps on both the plasma membrane and endoplasmic reticulum, which sequester [Ca 2+ ] i (Billups and Forsythe 2002; Chanaday and Kavalali 2022; Wan et al. 2012).”

“Syt1 is essential for optimal SV endocytosis, although its mechanism is still debated (Bolz et al. 2023; Yao, Kwon, et al. 2011).”

“For example, in mature nerve terminals, a series of v-SNARE proteins including Vti1a, VAMP4 and VAMP7 all appear to bias neurotransmitter release towards spontaneous SV fusion when overexpressed in primary neuronal cultures (Bal et al. 2013; Lin et al. 2020; Ramirez et al. 2012).”

“Rodents display a much earlier peak of synaptogenesis, which is typically maximal at the second to third postnatal week, although some recent publications report steady synapto-genesis until young adulthood (Semple et al. 2013; Wildenberg et al. 2023).”

“…by calcium influx, but whether the calcium influx is generated by VGCCs or other sources remains controversial, especially in glutamatergic synapses (Babiec and O'Dell 2018; Chanaday et al. 2021; Dai et al. 2015; Liu et al. 2018; Reese and Kavalali 2015; Williams and Smith 2018; Xu et al. 2009).”

“…its heavy or light chain has been shown to have either little impact (Kononenko et al. 2014; López-Hernández et al. 2022; Soykan et al. 2016) or retard the retrieval of various pHluorin reporters (López-Hernández et al. 2022; Lopez-Murcia et al. 2014; Nicholson-Fish et al. 2015; Wei et al. 2024).”

“The same experiment in layer 5 pyramidal neurons showed no significant difference in the replenishment rate of the RRP from P8–10 to P21–24 (Bornschein et al. 2020; Bornschein and Schmidt 2019).”

“Neurotransmitter is loaded into SVs against its concentration gradient by exploiting a pro-tonmotive force generated by a V-type ATPase located on the SV membrane (Gowrisankaran and Milosevic 2020; Pulido and Ryan 2021).”

“As mentioned earlier, this is triggered by the binding of free calcium ions to Syt1, which drives altered interactions with both the assembled SNARE complex and plasma membrane, resulting in the synchronisation of neurotransmitter release to neuronal activity (Hui et al. 2009; Toulmé et al. 2024).”

“…into the plasma membrane and becomes fluorescent) was dependent on calcium in 2- day-old Xenopus spinal cord neurons, and the internalisation of Syt1 antibodies was also dependent on the extracellular calcium concentration at DIV 2–4 in hippocampal neurons (Coco et al. 2002; Dai and Peng 1996).”

“GABAergic presynapses in dorsal D motoneurons in larval C. elegans that innervate ventral muscle cells are eliminated as part of the rewiring process (Mulcahy et al. 2022).”

“…(GABA)ergic (Briševac et al. 2021) Basket cell-Purkinje cell synapse in the cerebellar cortex presents a predominance of P/Q-type VGCCs throughout their development from P7–9 to P21–23, with minimal ωCgTx-GVIA sensitivity observed when the amplitude of evoked IPSCs was examined (Chen et al. 2024).”

“Furthermore, the recruitment of both clathrin and dynamin-1 was blocked by inhibition of the calcium and calmodulin-dependent protein phosphatase CaN, which activates dephosphins to trigger ADBE (Cousin and Robinson 2001; Tojima et al. 2010).”

“In primary cultures of medium spiny neurons from murine basal ganglia, which are mostly GABAergic, a similar increase in the amplitude of TTx-sensitive sIPSCs was observed from DIV 7–8 to DIV 12–14 (Arama et al. 2015).”

“UFE and ADBE may also share the same molecular mechanism, although they were originally proposed to be triggered by mild and intense stimulation respectively (Blumrich et al. 2023; Clayton and Cousin 2009; Imoto et al. 2022, 2024; Smillie et al. 2005; Watanabe et al. 2018; Xue et al. 2011).”

“Therefore, there appears to be a growing consensus that exocytosis becomes more tightly coupled to the calcium influx due to the closer physical organisation of VGCCs to the SV fusion machinery, with varying degrees depending on the types of synapses (Rebola et al. 2019).”

“An alternative hypothesis is that the high prevalence of spontaneous events is due to fusion of ‘constitutively releasing vesicles’, which deliver key molecules to the developing synapse and after multiple fusion events acquire a typical SV identity (Truckenbrodt and Rizzoli 2014).”

“In contrast, BAPTA was equally efficient in inhibiting EPSCs in both age groups (Fedchyshyn and Wang 2005).”

“UFE and ADBE may also share the same molecular mechanism, although they were originally proposed to be triggered by mild and intense stimulation respectively (Blumrich et al. 2023; Clayton and Cousin 2009; Imoto et al. 2022, 2024; Smillie et al. 2005; Watanabe et al. 2018; Xue et al. 2011).”

“With increasing maturity, this phenomenon tapers off around the second postnatal week in rodents (Blankenship and Feller 2010; Kersbergen and Bergles 2024; Luhmann et al. 2016; Pires et al. 2021).”

“With increasing maturity, this phenomenon tapers off around the second postnatal week in rodents (Blankenship and Feller 2010; Kersbergen and Bergles 2024; Luhmann et al. 2016; Pires et al. 2021).”

“For more extensive coverage of these specific topics, refer to other reviews (Leterrier 2018; Nozumi and Igarashi 2018; Petzoldt 2023; Rizalar et al. 2021).”

“The same experiment in layer 5 pyramidal neurons showed no significant difference in the replenishment rate of the RRP from P8–10 to P21–24 (Bornschein et al. 2020; Bornschein and Schmidt 2019).”

“This elimination appears to require many of the key molecules required for ADBE, including CaN, dynamin, syndapin, the Arp2/3 complex and Rab-11 (Cuentas-Condori et al. 2023).”

“…its heavy or light chain has been shown to have either little impact (Kononenko et al. 2014; López-Hernández et al. 2022; Soykan et al. 2016) or retard the retrieval of various pHluorin reporters (López-Hernández et al. 2022; Lopez-Murcia et al. 2014; Nicholson-Fish et al. 2015; Wei et al. 2024).”

“For more extensive coverage of these specific topics, refer to other reviews (Leterrier 2018; Nozumi and Igarashi 2018; Petzoldt 2023; Rizalar et al. 2021).”

“…the plasma membrane of the release site, while priming renders SVs fusion-competent by changing the conformation of the SNARE complex, a process coordinated via interactions with a series of molecules including Munc-13, Munc-18, complexin and Syt1 (An and Lindau 2024; Imig et al. 2014; Rizo 2022).”

“A key component of this is the delivery and tethering of VGCCs (Chin and Kaeser 2024; Held et al. 2020), with the latter requiring interactions with a series of cell adhesion molecules, such as the neurexins and laminins (Chen et al. 2011; Luo et al. 2020; Nishimune et al. 2004).”

“Finally, studies that examined the distance between Ca v 2.1 channels and the active zone protein Munc13 using the super-resolution microscopy technique 3D-STORM revealed a reduction in separation from P6 to 18 in lemniscal fibres in the ventral posterior medial nucleus (Midorikawa et al. 2024).”

“For example, the gamma-aminobutyric acid (GABA)ergic (Briševac et al. 2021) Basket cell-Purkinje cell synapse in the cerebellar cortex presents a predominance of P/Q-type VGCCs throughout their development from P7–9 to P21–23, with minimal ωCgTx-GVIA sensitivity observed when the amplitude of…”

“This process of synaptogenesis is then followed by a stationary phase, during which synapses mature (Kim et al. 2024).”

“Similarly, knockout of β-actin resulted in a dramatic increase in spontaneous SV fusion events (Wu et al. 2025) similar to previous interventions that depo-lymerise actin microfilaments (Morales et al. 2000).”

“The VGCC-dependence of miniature events in hippocampal neurons was supported by studies in both hippocampal autaptic cultures and at the Calyx of Held after hearing onset (P18) (Lee et al. 2022).”

“With increasing maturity, this phenomenon tapers off around the second postnatal week in rodents (Blankenship and Feller 2010; Kersbergen and Bergles 2024; Luhmann et al. 2016; Pires et al. 2021).”

“This process is particularly intense during childhood and adolescence but continues at a reduced pace throughout adulthood [13].”

“2) can be divided into several steps, including vesicle mobilization, docking, priming, fusion, and recycling (28).”