Immature Synapses Research Articles

Estimation of cumulative amplitude distributions of miniature postsynaptic currents allows characterising their multimodality, quantal size and variability

A miniature postsynaptic current (mPSC) is a small, rare, and highly variable spontaneous synaptic event that is generally caused by the spontaneous release of single vesicles. The amplitude and variability of mPSCs are key measures of the postsynaptic processes and are taken as the main characteristics of an elementary unit (quantal size) in traditional quantal analysis of synaptic transmission. Due to different sources of biological and measurement noise, recordings of mPSCs exhibit high trial-to-trial heterogeneity, and experimental measurements of mPSCs are usually noisy and scarce, making their analysis demanding. Here, we present a sequential procedure for precise analysis of mPSC amplitude distributions for the range of small currents. To illustrate the developed approach, we chose previously obtained experimental data on the effect of the extracellular matrix on synaptic plasticity. The proposed statistical technique allowed us to identify previously unnoticed additional modality in the mPSC amplitude distributions, indicating the formation of new immature synapses upon ECM attenuation. We show that our approach can reliably detect multimodality in the distributions of mPSC amplitude, allowing for accurate determination of the size and variability of the quantal synaptic response. Thus, the proposed method can significantly expand the informativeness of both existing and newly obtained experimental data. We also demonstrated that mPSC amplitudes around the threshold of microcurrent excitation follow the Gumbel distribution rather than the binomial statistics traditionally used for a wide range of currents, either for a single synapse or when taking into consideration small influences of the adjacent synapses. Such behaviour is argued to originate from the theory of extreme processes. Specifically, recorded mPSCs represent instant random current fluctuations, among which there are relatively larger spikes (extreme events). They required more level of coherence that can be provided by different mechanisms of network or system level activation including neuron circuit signalling and extrasynaptic processes.

Endogenous Cannabinoid Receptors Modulate Plasticity at Immature Synapses

To explore the mechanism of endocannabinoid cannabinoid receptor 1 (CB1) receptor pathway that regulates synaptic plasticity in the dorsal horn of the spinal cord of rats with neuropathic pain at different ages. Neonatal, juvenile, and adult male sprague dawley (SD) rats were divided into the spinal nerve preservation injury (SNI), SNI + Anandamide (AEA), SNI + D-AP5, SNI + CNQX, SNI + D-AP5 + AEA, SNI + CNQX + AEA, sham SNI, sham SNI + AEA, sham SNI + D-AP5, sham SNI + CNQX, sham SNI + D-AP5 + AEA, and sham SNI + CNQX + AEA groups, respectively. Paw withdrawal threshold (PWT) and long-term potentiation (LTP) of the spinal dorsal horn PS (field potential) were assessed to judge the spinal cord's functional state. Immunohistochemical staining and Western blot were conducted to detect CB1 protein levels in the spinal dorsal horn. The LTP response in the spinal cord was alleviated in the SNI + AEA group. After treatment with the N-methyl-D-aspartate (NMDA) receptor blocker D-AP5, the LTP of neonatal A nerve was relieved further. After treatment with the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor blocker CNQX, LTP change in the A nerve was not obvious. The LTP of the A and C nerves were relieved after D-AP5 or CNQX treatment in young and adult animals; however, the blocking effect of CNQX was obvious. The altered levels of PWT and CB1 support these results. The CB1 receptor activation produces analgesia in neonatal rats through NMDA receptor formation for PS inhibitory activity. In juvenile and adult rats, this phenomenon was effectuated through NMDA and AMPA receptors. This difference could be attributed to the varied number of NMDA and/or AMPA receptors activated during development and changes in the NMDA/AMPA receptor ratio.

Voltage dependent allosteric modulation of IPSCs by benzodiazepines

GABAA receptors (GABAAR) are inhibitory ion channels ubiquitously expressed in the central nervous system and play critical roles in brain development and function. Benzodiazepines are positive allosteric modulators of GABAAR, enhancing channel opening frequency when GABA is bound to the receptor. Midazolam is a commonly used benzodiazepine. It is frequently used for premature infants, but the long-term consequences of its use in this patient population are not well established. Here, we studied the acute effects of midazolam on immature synapses. Using a rodent organotypic hippocampal slice preparation, we evaluated how midazolam affects inhibitory synaptic transmission onto CA1 pyramidal neurons. We found that 1 μM midazolam enhances evoked inhibitory post synaptic currents (eIPSCs) at a holding potential of −60 mV. Similarly, 1 μM midazolam enhances miniature IPSCs (mIPSCs) in CA1 pyramidal neurons at holding potentials of −60 mV and −30 mV. At depolarized holding potentials, however, midazolam no longer enhances mIPSCs. Depolarization of the postsynaptic cell by itself increases mIPSC decay, which occludes the allosteric effects of midazolam. These results provide insight into how a benzodiazepine and membrane voltage may modulate GABAAR function in developing circuits.

Presynapse Formation Assay Using Presynapse Organizer Beads and “Neuron Ball” Culture

During neuronal development, synapse formation is an important step to establish neural circuits. To form synapses, synaptic proteins must be supplied in appropriate order by transport from cell bodies and/or local translation in immature synapses. However, it is not fully understood how synaptic proteins accumulate in synapses in proper order. Here, we present a novel method to analyze presynaptic formation by using the combination of neuron ball culture with beads to induce presynapse formation. Neuron balls that is neuronal cell aggregates provide axonal sheets far from cell bodies and dendrites, so that weak fluorescent signals of presynapses can be detected by avoiding overwhelming signals of cell bodies. As beads to trigger presynapse formation, we use beads conjugated with leucine-rich repeat transmembrane neuronal 2 (LRRTM2), an excitatory presynaptic organizer. Using this method, we demonstrated that vesicular glutamate transporter 1 (vGlut1), a synaptic vesicle protein, accumulated in presynapses faster than Munc18-1, an active zone protein. Munc18-1 accumulated translation-dependently in presynapse even after removing cell bodies. This finding indicates the Munc18-1 accumulation by local translation in axons, not transport from cell bodies. In conclusion, this method is suitable to analyze accumulation of synaptic proteins in presynapses and source of synaptic proteins. As neuron ball culture is simple and it is not necessary to use special apparatus, this method could be applicable to other experimental platforms.

Immature Synaptic Contacts in Human Congenital Hydrocephalus. An Electron Microscopic study

Aggelopoulos et al. [1] analyzed the synaptogenesis in the dorsal lateral geniculate nucleus of the rat and demonstrated that during the first stage, which spans the first 4 days of life, there are only a few immature synapses scattered throughout the nucleus.

Astrocyte-Secreted Chordin-like 1 Drives Synapse Maturation and Limits Plasticity by Increasing Synaptic GluA2 AMPA Receptors

In the developing brain, immature synapses contain calcium-permeable AMPA glutamate receptors (AMPARs) that are subsequently replaced with GluA2-containing calcium-impermeable AMPARs as synapses stabilize and mature. Here, we show that this essential switch in AMPARs and neuronal synapse maturation is regulated by astrocytes. Using biochemical fractionation of astrocyte-secreted proteins and mass spectrometry, we identified that astrocyte-secreted chordin-like 1 (Chrdl1) is necessary and sufficient to induce mature GluA2-containing synapses to form. This function of Chrdl1 is independent of its role as an antagonist of bone morphogenetic proteins (BMPs). Chrdl1 expression is restricted to cortical astrocytes invivo, peaking at the time of the AMPAR switch. Chrdl1 knockout (KO) mice display reduced synaptic GluA2 AMPARs, altered kinetics of synaptic events, and enhanced remodeling in an invivo plasticity assay. Studies have shown that humans with mutations in Chrdl1 display enhanced learning. Thus astrocytes, via the release of Chrdl1, promote GluA2-dependent synapse maturation and thereby limit synaptic plasticity.

Synapse-selective rapid potentiation of hippocampal synaptic transmission by 7,8-dihydroxyflavone.

AimThe identification of 7,8‐dihydroxyflavone (DHF) as a small molecule agonist for tropomyosin‐related kinase B (TrkB) facilitated understanding of the role of TrkB signaling in regulating higher brain functions. DHF can penetrate the blood‐brain barrier after systemic administration and changes the performance of cognitive and emotional behavioral tasks. However, it is poorly understood how DHF modulates neuronal functions at cellular levels. Aiming to understand the cellular basis underlying DHF‐induced modifications of the brain functions, we examined the effects of DHF on the hippocampal excitatory synaptic transmission.MethodsField excitatory postsynaptic potentials were recorded using hippocampal slices prepared from adult male mice. Effects of bath‐applied DHF on the synaptic efficacy were examined.ResultsWe found that DHF induced robust synaptic potentiation at the mossy fiber to CA3 synapse. DHF had minimal effects at other hippocampal excitatory synapses or at immature mossy fiber synapse in juvenile mice. The TrkB receptor blockers K252a and ANA‐12 did not affect the DHF‐induced synaptic potentiation. Drug screening revealed that relatively low concentrations of 2‐aminoethoxydiphenylborane blocked the DHF‐induced synaptic potentiation.ConclusionOur results demonstrate that DHF selectively potentiates hippocampal mossy fiber synaptic transmission via a TrkB receptor‐independent mechanism. This novel neuromodulatory effect of DHF may influence higher brain functions by itself or together with the activation of the TrkB receptor. The rapid induction of the potentiation implies its potential importance in the acute behavioral effects of DHF.

Mechanically patterned neuromuscular junctions-in-a-dish have improved functional maturation.

Motor neuron (MN) diseases are progressive disorders resulting from degeneration of neuromuscular junctions (NMJs), which form the connection between MNs and muscle fibers. NMJ-in-a-dish models have been developed to examine human MN-associated dysfunction with disease; however such coculture models have randomly oriented myotubes with immature synapses that contract asynchronously. Mechanically patterned (MP) extracellular matrix with alternating soft and stiff stripes improves current NMJ-in-a-dish models by inducing both mouse and human myoblast durotaxis to stripes where they aligned, differentiated, and fused into patterned myotubes. Compared to conventional culture on rigid substrates or unpatterned hydrogels, MP substrates supported increased differentiation and fusion, significantly larger acetylcholine (ACh) receptor clusters, and increased expression of MuSK and Lrp4, two cell surface receptors required for NMJ formation. Robust contractions were observed when mouse myotubes were stimulated by ACh, with twitch duration and frequency most closely resembling those for mature muscle on MP substrates. Fused myotubes, when cocultured with MNs, were able to form even larger NMJs. Thus MP matrices produce more functionally active NMJs-in-a-dish, which could be used to elucidate disease pathology and facilitate drug discovery.

Astrocyte lipid metabolism is critical for synapse development and function in vivo.

The brain is considered to be autonomous in lipid synthesis with astrocytes producing lipids far more efficiently than neurons. Accordingly, it is generally assumed that astrocyte-derived lipids are taken up by neurons to support synapse formation and function. Initial confirmation of this assumption has been obtained in cell cultures, but whether astrocyte-derived lipids support synapses in vivo is not known. Here, we address this issue and determined the role of astrocyte lipid metabolism in hippocampal synapse formation and function in vivo. Hippocampal protein expression for the sterol regulatory element-binding protein (SREBP) and its target gene fatty acid synthase (Fasn) was found in astrocytes but not in neurons. Diminishing SREBP activity in astrocytes using mice in which the SREBP cleavage-activating protein (SCAP) was deleted from GFAP-expressing cells resulted in decreased cholesterol and phospholipid secretion by astrocytes. Interestingly, SCAP mutant mice showed more immature synapses, lower presynaptic protein SNAP-25 levels as well as reduced numbers of synaptic vesicles, indicating impaired development of the presynaptic terminal. Accordingly, hippocampal short-term and long-term synaptic plasticity were defective in mutant mice. These findings establish a critical role for astrocyte lipid metabolism in presynaptic terminal development and function in vivo. GLIA 2017;65:670-682.

Altered thalamocortical development in the SAP102 knockout model of intellectual disability.

Genetic mutations known to cause intellectual disabilities (IDs) are concentrated in specific sets of genes including both those encoding synaptic proteins and those expressed during early development. We have characterized the effect of genetic deletion of Dlg3, an ID-related gene encoding the synaptic NMDA-receptor interacting protein synapse-associated protein 102 (SAP102), on development of the mouse somatosensory cortex. SAP102 is the main representative of the PSD-95 family of postsynaptic MAGUK proteins during early development and is proposed to play a role in stabilizing receptors at immature synapses. Genetic deletion of SAP102 caused a reduction in the total number of thalamocortical (TC) axons innervating the somatosensory cortex, but did not affect the segregation of barrels. On a synaptic level SAP102 knockout mice display a transient speeding of NMDA receptor kinetics during the critical period for TC plasticity, despite no reduction in GluN2B-mediated component of synaptic transmission. These data indicated an interesting dissociation between receptor kinetics and NMDA subunit expression. Following the critical period NMDA receptor function was unaffected by loss of SAP102 but there was a reduction in the divergence of TC connectivity. These data suggest that changes in synaptic function early in development caused by mutations in SAP102 result in changes in network connectivity later in life.

Chronic Social Stress Affects Synaptic Maturation of Newly Generated Neurons in the Adult Mouse Dentate Gyrus.

Background:Chronic stress has been found to suppress adult neurogenesis, but it remains unclear whether it may affect the maturation process of adult-born neurons. Here, we examined the influence of chronic social defeat stress on the morphological and electrophysiological properties of adult-born dentate granule cells at different developmental stages.Methods:Adult C57BL/6 mice were subjected to 10 days of chronic social defeat stress followed by a social interaction test 24 hours after the last defeat. Defeated mice were segregated into susceptible and unsusceptible subpopulations based on a measure of social interaction test. Combining electrophysiology with retrovirus-mediated birth-dating and labeling, we examined the impact of chronic social defeat stress on temporal regulation of synaptic plasticity of adult-born dentate granule cells along their maturation.Results:Chronic social defeat stress decreases the survival and dendritic complexity of adult-born dentate granule cells. While chronic social defeat stress doesn’t alter the intrinsic electrophysiological properties and synaptic transmission of surviving adult-born dentate granule cells, it promotes the developmental switch in synaptic N-methyl-D-aspartate receptors from predominant GluN2B- to GluN2A-containing receptors, which transform the immature synapse of adult-born dentate granule cells from one that exhibits enhanced long-term potentiation to one that has normal levels of long-term potentiation. Furthermore, chronic social defeat stress increases the level of endogenous repressor element-1 silencing transcription factor mRNA in adult-born dentate granule cells, and knockdown of the repressor element-1 silencing transcription factor in adult-born dentate granule cells rescues chronic social defeat stress-induced morphological deficits and accelerated developmental switch in synaptic N-methyl-D-aspartate receptor subunit composition.Conclusions:These results uncover a previously unsuspected role of chronic social defeat stress in regulating adult neurogenesis and suggest that chronic social defeat stress can affect synaptic maturation process of adult-born dentate granule cells.

The LGI1–ADAM22 protein complex directs synapse maturation through regulation of PSD-95 function

Synapse development is coordinated by a number of transmembrane and secreted proteins that come together to form synaptic organizing complexes. Whereas a variety of synaptogenic proteins have been characterized, much less is understood about the molecular networks that support the maintenance and functional maturation of nascent synapses. Here, we demonstrate that leucine-rich, glioma-inactivated protein 1 (LGI1), a secreted protein previously shown to modulate synaptic AMPA receptors, is a paracrine signal released from pre- and postsynaptic neurons that acts specifically through a disintegrin and metalloproteinase protein 22 (ADAM22) to set postsynaptic strength. We go on to describe a novel role for ADAM22 in maintaining excitatory synapses through PSD-95/Dlg1/zo-1 (PDZ) domain interactions. Finally, we show that in the absence of LGI1, the mature synapse scaffolding protein PSD-95, but not the immature synapse scaffolding protein SAP102, is unable to modulate synaptic transmission. These results indicate that LGI1 and ADAM22 form an essential synaptic organizing complex that coordinates the maturation of excitatory synapses by regulating the functional incorporation of PSD-95.

Temporal dynamics in an immunological synapse: Role of thermal fluctuations in signaling.

The article analyzes the contribution of stochastic thermal fluctuations in the attachment times of the immature T-cell receptor TCR: peptide-major-histocompatibility-complex pMHC immunological synapse bond. The key question addressed here is the following: how does a synapse bond remain stabilized in the presence of high-frequency thermal noise that potentially equates to a strong detaching force? Focusing on the average time persistence of an immature synapse, we show that the high-frequency nodes accompanying large fluctuations are counterbalanced by low-frequency nodes that evolve over longer time periods, eventually leading to signaling of the immunological synapse bond primarily decided by nodes of the latter type. Our analysis shows that such a counterintuitive behavior could be easily explained from the fact that the survival probability distribution is governed by two distinct phases, corresponding to two separate time exponents, for the two different time regimes. The relatively shorter timescales correspond to the cohesion:adhesion induced immature bond formation whereas the larger time reciprocates the association:dissociation regime leading to TCR:pMHC signaling. From an estimate of the bond survival probability, we show that, at shorter timescales, this probability P(Δ)(τ) scales with time τ as a universal function of a rescaled noise amplitude D/Δ(2), such that P(Δ)(τ)∼τ(-(Δ/√[D]+1/2)),Δ being the distance from the mean intermembrane (T cell:Antigen Presenting Cell) separation distance. The crossover from this shorter to a longer time regime leads to a universality in the dynamics, at which point the survival probability shows a different power-law scaling compared to the one at shorter timescales. In biological terms, such a crossover indicates that the TCR:pMHC bond has a survival probability with a slower decay rate than the longer LFA-1:ICAM-1 bond justifying its stability.

Activity-dependent upregulation of presynaptic kainate receptors at immature CA3-CA1 synapses.

Presynaptic kainate-type glutamate receptors (KARs) regulate glutamate release probability and short-term plasticity in various areas of the brain. Here we show that long-term depression (LTD) in the area CA1 of neonatal rodent hippocampus is associated with an upregulation of tonic inhibitory KAR activity, which contributes to synaptic depression and causes a pronounced increase in short-term facilitation of transmission. This increased KAR function was mediated by high-affinity receptors and required activation of NMDA receptors, nitric oxide (NO) synthetase, and postsynaptic calcium signaling. In contrast, KAR activity was irreversibly downregulated in response to induction of long-term potentiation in a manner that depended on activation of the TrkB-receptor of BDNF. Both tonic KAR activity and its plasticity were restricted to early stages of synapse development and were lost in parallel with maturation of the network due to ongoing BDNF-TrkB signaling. These data show that presynaptic KARs are targets for activity-dependent modulation via diffusible messengers NO and BDNF, which enhance and depress tonic KAR activity at immature synapses, respectively. The plasticity of presynaptic KARs in the developing network allows nascent synapses to shape their response to incoming activity. In particular, upregulation of KAR function after LTD allows the synapse to preferentially pass high-frequency afferent activity. This can provide a potential rescue from synapse elimination by uncorrelated activity and also increase the computational dynamics of the developing CA3-CA1 circuitry.

VGLUT3 does not synergize GABA/glycine release during functional refinement of an inhibitory auditory circuit.

The vesicular glutamate transporter 3 (VGLUT3) is expressed at several locations not normally associated with glutamate release. Although the function of this protein has been generally elusive, when expressed in non-glutamatergic synaptic terminals, VGLUT3 can not only allow glutamate co-transmission but also synergize the action of non-glutamate vesicular transporters. Interestingly, in the immature glycinergic projection between the medial nucleus of the trapezoid body (MNTB) and the lateral superior olive (LSO) of auditory brainstem, the transient early expression of VGLUT3 is required for normal developmental refinement. It has however been unknown whether the primary function of VGLUT3 in development of these inhibitory synapses is to enable glutamate release or to promote loading of inhibitory neurotransmitter through vesicular synergy. Using tissue from young mice in which Vglut3 had been genetically deleted, we evaluated inhibitory neurotransmission in the MNTB-LSO pathway. Our results show, in contrast to what has been seen at adult synapses, that VGLUT3 expression has little or no effect on vesicular synergy at the immature glycinergic synapse of brainstem. This finding supports the model that the primary function of increased VGLUT3 expression in the immature auditory brainstem is to enable glutamate release in a developing inhibitory circuit.

Alterations of the synapse of the inner retinal layers after chronic intraocular pressure elevation in glaucoma animal model

BackgroundDendrites of retinal ganglion cells (RGCs) synapse with axon terminals of bipolar cells in the inner plexiform layer (IPL). Changes in RGC dendrites and synapses between bipolar cells in the inner retinal layer may critically alter the function of RGCs in glaucoma. Recently, synaptic plasticity has been observed in the adult central nervous system, including the outer retinal layers. However, few studies have focused on changes in the synapses between RGCs and bipolar cells in glaucoma. In the present study, we used a rat model of ocular hypertension induced by episcleral vein cauterization to investigate changes in synaptic structure and protein expression in the inner retinal layer at various time points after moderate intraocular pressure (IOP) elevation.ResultsSynaptophysin, a presynaptic vesicle protein, increased throughout the IPL, outer plexiform layer, and outer nuclear layer after IOP elevation. Increased synaptophysin after IOP elevation was expressed in bipolar cells in the innermost IPL. The RGC marker, SMI-32, co-localized with synaptophysin in RGC dendrites and were significantly increased at 1 week and 4 weeks after IOP elevation. Both synaptophysin and postsynaptic vesicle protein, PSD-95, were increased after IOP elevation by western blot analysis. Ribbon synapses in the IPL were quantified and structurally evaluated in retinal sections by transmission electron microscopy. After IOP elevation the total number of ribbon synapses decreased. There were increases in synapse diameter and synaptic vesicle number and decreases in active zone length and the number of docked vesicles after IOP elevation.ConclusionsAlthough the total number of synapses decreased as RGCs were lost after IOP elevation, there are attempts to increase synaptic vesicle proteins and immature synapse formation between RGCs and bipolar cells in the inner retinal layers after glaucoma induction.

Maturation of silent synapses in amygdala-accumbens projection contributes to incubation of cocaine craving

In rat models of drug relapse and craving, cue-induced cocaine seeking progressively increases after drug withdrawal. This ‘incubation of cocaine craving’ is partially mediated by time-dependent adaptations at glutamatergic synapses in nucleus accumbens. However, the circuit-level adaptations mediating this plasticity remain elusive. Here we studied silent synapses—often regarded as immature synapses that express stable NMDA receptors with AMPA receptors either absent or labile—in basolateral amygdala-to-accumbens projection in incubation of cocaine craving. Silent synapses were detected within this projection during early withdrawal from cocaine. As the withdrawal period progressed, these silent synapses became ‘unsilenced’, a process involving synaptic insertion of calcium-permeable AMPA receptors (CP-AMPARs). In vivo optogenetic stimulation-induced downregulation of CP-AMPARs at amygdala-to-NAc synapses, which re-silenced some of the previously silent synapses after prolonged withdrawal, decreased cocaine incubation. Our finding indicates that silent synapse-based reorganization of the amygdala-to-accumbens projection is critical for persistent cocaine craving and relapse after withdrawal.

Adenosine A1 receptor activation mediates the developmental shift at layer 5 pyramidal cell synapses and is a determinant of mature synaptic strength

During the first postnatal month glutamatergic synapses between layer 5 pyramidal cells in the rodent neocortex switch from an immature state exhibiting a high probability of neurotransmitter release, large unitary amplitude and synaptic depression to a mature state with decreased probability of release, smaller unitary amplitude and synaptic facilitation. Using paired recordings, we demonstrate that the developmental shift in release probability at synapses between rat somatosensory layer 5 thick-tufted pyramidal cells is mediated by a higher and more heterogeneous activation of presynaptic adenosine A1 receptors. Immature synapses under control conditions exhibited distributions of coefficient of variation, failure rate and release probability that were almost coincident with the A1 receptor blocked condition; however, mature synapses under control conditions exhibited much broader distributions that spanned those of both the A1 receptor agonized and antagonized conditions. Immature and mature synapses expressed A1 receptors with no observable difference in functional efficacy and therefore the heterogeneous A1 receptor activation seen in the mature neocortex appears due to increased adenosine concentrations that vary between synapses. Given the central role demonstrated for A1 receptor activation in determining synaptic amplitude and the statistics of transmission between mature layer 5 pyramidal cells, the emplacement of adenosine sources and sinks near the synaptic terminal could constitute a novel form of long-term synaptic plasticity.

Developmental upregulation of presynaptic NCKX underlies the decrease of mitochondria-dependent posttetanic potentiation at the rat calyx of Held synapse

The sensitivity of posttetanic potentiation (PTP) to high-frequency stimulation (HFS) steeply decays during the first 2 postnatal weeks. We investigated the underlying mechanisms for the developmental change of PTP induced by HFS (100 Hz, 2 s) at postnatal days 4-6 and 9-11 at the rat calyx of Held synapse. Low-concentration tetraphenylphosphonium (2 μM), an inhibitor of mitochondrial Na(+)/Ca(2+) exchanger, suppressed the amount of posttetanic residual Ca(2+) and PTP to a larger extent at the immature calyx synapse, indicating a developmental reduction of mitochondrial contribution to PTP. The higher amount of mitochondrial Ca(2+) uptake during HFS and consequent posttetanic residual Ca(2+) at the immature calyx of Held was associated with higher peak of HFS-induced Ca(2+) transients, most likely because the mitochondrial Ca(2+) uptake during HFS was supralinearly dependent on the presynaptic resting Ca(2+) level. Probing into the contribution of Na(+)/Ca(2+) exchangers to Ca(2+) clearance, we found a specific upregulation of the K(+)-dependent Na(+)/Ca(2+) exchanger (NCKX) activity in the mature calyx of Held. We conclude that the upregulation of NCKX limits the Ca(2+) buildup and inhibits mitochondrial Ca(2+) uptake during HFS, which in turn results in the reduction of posttetanic residual Ca(2+) and PTP at the mature calyx of Held synapse.

Mechanisms underlying induction of LTP-associated changes in short-term dynamics of transmission at immature synapses

While the activity-dependent mechanisms guiding functional maturation of synaptic transmission postsynaptically are well characterized, less is known about the corresponding presynaptic mechanisms. Here we show that during the first postnatal week, a subset of CA3–CA1 synapses express postsynaptically induced LTP that is tightly associated with a robust decrease in synaptic facilitation, consistent with an increase in release probability (Pr). The loss of facilitation is readily induced by physiologically relevant pairing protocols at immature synapses and is dependent on activation of NMDA-receptors but not L-type calcium channels. The putative pre- and postsynaptic components of neonatal LTP were distinguished in their downstream signaling requirements, PKC activity being selectively needed for the decrease in facilitation but not for synaptic potentiation per se. These data suggest that maturation of glutamatergic synapses involves a critical period during which presynaptic function is highly susceptible to activity-dependent regulation via a PKC-dependent mechanism.