Time and exposure to serotonin affect releasability of recycled vesicles at crayfish claw opener muscle synapses

The crayfish claw opener neuromuscular junction is a biological model for studying presynaptic neuromodulation by serotonin (5HT) and synaptic vesicle recycling. It has been hypothesized that 5HT enhances release by recruiting a population of either previously nonrecycling or “reluctant” vesicles to increase the readily releasable pool. To determine if 5HT activates a distinct population of synaptic vesicles, recycling membranes were labeled with the membrane dye, FM1-43. Unloading (destaining) protocols could not resolve a population of vesicles that were only releasable in the presence of 5HT. Instead, we conclude synaptic vesicles change behavior in axon terminals independent of 5HT, becoming less likely to exocytose and unload dye over periods of >1 hr after recycling. We hypothesized this to be due to the slow conversion of a portion of recycled vesicles to a difficult to release state. The possibility that vesicles in these pools were spatially separated within the terminal was tested using photoconversion of FM1-43 and transmission electron microscopy. The location of FM1-43-labeled vesicles fixed 2 min following 3 min of 20-Hz stimulation did not reveal preferential localization of recycling vesicles specifically near release sites and the distribution of labeled vesicles was not significantly different between early (2 min) and late (180 min) time points. Terminals fixed 30 s following stimulation contained a significant proportion of vesicular structures equivalent in diameter to 2–5 regular vesicles, with multivesicular bodies and calveoli rarely seen, suggesting that endocytosis during sustained release at crayfish terminals occurs via multiple routes, most commonly through large “vesicle” intermediates.     

Impairment of catecholamine systems during induction of long-term potentiation at hippocampal CA1 synapses in HPC-1/syntaxin 1A knock-out mice

The membrane protein HPC-1/syntaxin 1A is believed to play a key role in synaptic vesicle exocytosis, and it was recently suggested to be required for synaptic plasticity. Despite evidence for the function of HPC-1/syntaxin 1A in synaptic plasticity, the underlying cellular mechanism is unclear. We found that although fast synaptic transmission and long-term depression were unaffected, HPC-1/syntaxin 1A knock-out (STX1A(-/-)) mice showed impaired long-term potentiation (LTP) in response to theta-burst stimulation in CA1 hippocampal slices. The impairment in LTP was rescued by the application of forskolin, an adenylyl cyclase activator, or more robust stimulation, suggesting that cAMP/protein kinase A signaling was suppressed in these mice. In addition, catecholamine release from the hippocampus was significantly reduced in STX1A(-/-) mice. Because HPC-1/syntaxin 1A regulates exocytosis of dense-core synaptic vesicles, which contain neuromodulatory transmitters such as noradrenaline, dopamine and 5-HT, we examined the effect of neuromodulatory transmitters on LTP induction. Noradrenaline and dopamine enhanced LTP induction in STX1A(-/-) mice, whereas catecholamine depletion reduced LTP induction in wild-type mice. Theses results suggest that HPC-1/syntaxin 1A regulates catecholaminergic systems via exocytosis of dense-core synaptic vesicles, and that deletion of HPC-1/syntaxin 1A causes impairment of LTP induction.     

Limited numbers of recycling vesicles in small CNS nerve terminals: implications for neural signaling and vesicular cycling.

The tiny nerve terminals of central synapses contain far fewer vesicles than preparations commonly used for analysis of neurosecretion. Photoconversion of vesicles rendered fluorescent with the dye FM1-43 directly identified vesicles capable of engaging in exo-endocytotic recycling following stimulated Ca(2+) entry. This recycling pool typically contained 30-45 vesicles, only a minority fraction (15-20% on average) of the total vesicle population. The smallness of the recycling pool would severely constrain rates of quantal neurotransmission if classical pathways were solely responsible for vesicle recycling. Fortunately, vesicles can undergo rapid retrieval and reuse in addition to conventional slow recycling, to the benefit of synaptic information flow and neuronal signaling.     

L-type calcium channels are involved in fast endocytosis at the mouse neuromuscular junction

We used fluorescence microscopy of FM dyes-labeled synaptic vesicles and electrophysiological recordings to examine the functional characteristics of vesicle recycling and study how different types of voltage-dependent Ca²⁺ channels (VDCCs) regulate the coupling of exocytosis and endocytosis at mouse neuromuscular junction.
Our results demonstrate the presence of at least two different pools of recycling vesicles: a high-probability release pool (i.e. a fast destaining vesicle pool), which is preferentially loaded during the first 5 s (250 action potentials) at 50 Hz; and a low-probability release pool (i.e. a slow destaining vesicle pool), which is loaded during prolonged stimulation and keeps on refilling after end of stimulation.
Our results suggest that a fast recycling pool mediates neurotransmitter release when vesicle use is minimal (i.e. during brief high-frequency stimulation), while vesicle mobilization from a reserve pool is the prevailing mechanism when the level of synaptic activity increases.
We observed that specific N- and L -type VDCC blockers had no effect on evoked transmitter release upon low-frequency stimulation (5 Hz). However, at high-frequency stimulation (50 Hz), L -type Ca²⁺ channel blocker increased FM2-10 destaining and at the same time diminished quantal release. Furthermore, when L -type channels were blocked, FM2-10 loading during stimulation was diminished, while the amount of endocytosis after stimulation was increased.
Our experiments suggest that L -type VDCCs promote endocytosis of synaptic vesicles, directing the newly formed vesicles to a high-probability release pool where they compete against unused vesicles.     

Role of the cAMP cascade in the turnover of synaptic vesicles of the frog motor nerve terminal

In experiments with frog neuromuscular preparations we have shown with the use of electrophysiological (two-electrode voltage clamp) and optical (fluorescent exocytotic dye FM1-43) techniques that during high-frequency stimulation (20 imp/s, 3 min) the cyclic adenosine monophosphate (cAMP) system had a complex effect on the exo-endocytotic cycle of synaptic vesicles. The activation of cAMP-dependent enzymes (100 μM 8-Br-cAMP, 50 μM Bt₂-cAMP) was accompanied by facilitation of exocytosis of the vesicles from a ready-to-release pool and enhancement of the endocytosis of synaptic vesicles. However, transport of the vesicles from the mobilization pool to the ready-to-release pool was disturbed and transmitter release was supported by the vesicles from the reserve pool. Blockage of adenylate cyclase (1 μM MDL) suppressed exocytosis of the vesicles from the ready-to-release pool, hindered replenishment of this pool with vesicles from the mobilization and reserve pools, and impaired endocytosis. Thus, stimulation of the cAMP pathway promotes vesicle recycling via a slow pathway and maintenance of transmitter release during high-frequency activity via vesicles from the reserve pool, whereas the background activity of adenylate cyclase is necessary for the effective development of all the key stages of the vesicular cycle.     

Membrane cholesterol regulates different modes of synaptic vesicle release and retrieval at the frog neuromuscular junction

We investigated the effects of cholesterol removal on spontaneous and KCl‐evoked synaptic vesicle recycling at the frog neuromuscular junction. Cholesterol removal by methyl‐β‐cyclodextrin (MβCD) induced an increase in the frequency of miniature end‐plate potentials (MEPPs) and spontaneous destaining of synaptic vesicles labeled with the styryl dye FM1‐43. Treatment with MβCD also increased the size of MEPPs without causing significant changes in nicotinic receptor clustering. At the ultrastructural level, synaptic vesicles from nerve terminals treated with MβCD were larger than those from control. In addition, treatment with MβCD reduced the fusion of synaptic vesicles that are mobilized during KCl‐evoked stimulation, but induced recycling of those vesicles that fuse spontaneously. We therefore suggest that MβCD might favor the release of vesicles that belong to a pool that is different from that involved in the KCl‐evoked release. These results reveal fundamental differences in the synaptic vesicle cycle for spontaneous and evoked release, and suggest that deregulation of cholesterol affects synaptic vesicle biogenesis and increases transmitter packing.     

Adenosine drives recycled vesicles to a slow‐release pool at the mouse neuromuscular junction

The effects of adenosine on neurotransmission have been widely studied by monitoring transmitter release. However, the effects of adenosine on vesicle recycling are still unknown. We used fluorescence microscopy of FM2‐10‐labeled synaptic vesicles in combination with intracellular recordings to examine whether adenosine regulates vesicle recycling during high‐frequency stimulation at mouse neuromuscular junctions. The A₁ adenosine receptor antagonist (8‐cyclopentyl‐1,3‐dipropylxanthine) increased the quantal content released during the first endplate potential, suggesting that vesicle exocytosis can be restricted by endogenous adenosine, which accordingly decreases the size of the recycling vesicle pool. Staining protocols designed to label specific vesicle pools that differ in their kinetics of release showed that all vesicles retrieved in the presence of 8‐cyclopentyl‐1,3‐dipropylxanthine were recycled towards the fast‐release pool, favoring its loading with FM2‐10 and suggesting that endogenous adenosine promotes vesicle recycling towards the slow‐release pool. In accordance with this effect, exogenous applied adenosine prevented the replenishment of the fast‐release vesicle pool and, thus, hindered its loading with the dye. We had found that, during high‐frequency stimulation, Ca²⁺ influx through L‐type channels directs newly formed vesicles to a fast‐release pool (Perissinotti et al., 2008). We demonstrated that adenosine did not prevent the effect of the L‐type blocker on transmitter release. Therefore, activation of the A₁ receptor promotes vesicle recycling towards the slow‐release pool without a direct effect on the L‐type channel. Further studies are necessary to elucidate the molecular mechanisms involved in the regulation of vesicle recycling by adenosine.     

Presynaptic ultrastructural correlates of long-term potentiation in the CA1 subfield of the hippocampus

To determine if long-term potentiation (LTP) is accompanied by changes in the ultrastructural distribution of calcium within presynaptic terminals, calcium was localized at the electron microscopic level using an oxalate/pyroantimonate histochemical technique. Following the induction of LTP at the Schaffer collateral/commissural synapses in the CA1 subfield of the rat hippocampal slice, there was a significant decrease (30%) in the percentage of synaptic vesicles containing calcium deposits. This effect could be accounted for by both a significant reduction in the average number of calcium deposit-bearing vesicles and a significant increase in the average number of synaptic vesicles per terminal profile in slices that displayed LTP. These changes persisted for at least one hour following the induction of LTP and were not observed in slices that received high-frequency stimulation in the presence of the N-methyl-D-aspartate (NMDA) receptor antagonist, 2-amino-5-phosphonovaleric acid (APV, 50 microM), which blocked LTP. These data suggest that LTP may be accompanied by long-term changes in intraterminal calcium homeostasis and the number of synaptic vesicles. These effects may be related to the reported increase in transmitter release following the induction of LTP.     

Correlation of miniature synaptic activity and evoked release probability in cultures of cortical neurons.

Spontaneous miniature synaptic activity is caused by action potential (AP)-independent release of transmitter vesicles and is regulated at the level of single synapses. In cultured cortical neurons we have used this spontaneous vesicle turnover to load the styryl dye FM1-43 into synapses with high rates of miniature synaptic activity. Automated selection procedures restricted analysis to synapses with sufficient levels of miniature activity-mediated FM1-43 uptake. After FM1-43 loading, vesicular FM1-43 release in response to AP stimulation was recorded at single synapses as a measure of release probability. We find that synapses with high rates of miniature activity possess significantly enhanced evoked release rates compared with a control population. Because the difference in release rates between the two populations is [Ca(2+)](o)-dependent, it is most likely caused by a difference in release probability. Within the subpopulation of synapses with high miniature activity, we find that the probabilities for miniature and AP-evoked release are correlated at single synaptic sites. Furthermore, the degree of miniature synaptic activity is correlated with the vesicle pool size. These findings suggest that both evoked and miniature vesicular release are regulated in parallel and that the frequency of miniature synaptic activity can be used as an indicator for evoked release efficacy.     

The optically determined size of exo/endo cycling vesicle pool correlates with the quantal content at the neuromuscular junction of Drosophila larvae.

According to the current theory of synaptic transmission, the amplitude of evoked synaptic potentials correlates with the number of synaptic vesicles released at the presynaptic terminals. Synaptic vesicles in presynaptic boutons constitute two distinct pools, namely, exo/endo cycling and reserve pools (). We defined the vesicles that were endocytosed and exocytosed during high K+ stimulation as the exo/endo cycling vesicle pool. To determine the role of exo/endo cycling vesicle pool in synaptic transmission, we estimated the quantal content electrophysiologically, whereas the pool size was determined optically using fluorescent dye FM1-43. We then manipulated the size of the pool with following treatments. First, to change the state of boutons of nerve terminals, motoneuronal axons were severed. With this treatment, the size of exo/endo cycling vesicle pool decreased together with the quantal content. Second, we promoted the FM1-43 uptake using cyclosporin A, which inhibits calcineurin activities and enhances endocytosis. Cyclosporin A increased the total uptake of FM1-43, but neither the size of exo/endo cycling vesicle pool nor the quantal content changed. Third, we increased the size of exo/endo cycling vesicle pool by forskolin, which enhances synaptic transmission. The forskolin treatment increased both the size of exo/endo cycling vesicle pool and the quantal content. Thus, we found that the quantal content was closely correlated with the size of exo/endo cycling vesicle pool but not necessarily with the total uptake of FM1-43 fluorescence by boutons. The results suggest that vesicles in the exo/endo cycling pool primarily participate in evoked exocytosis of vesicles.     

The role of nitric oxide in the regulation of neurotransmitter release and processes of exo- and endocytosis of synaptic vesicles in mouse motor nerve endings

In experiments with mouse diaphragm muscle, the effects of a nitric oxide (NO) donor, S-nitroso- N-acetyl-DL-penicillamine (SNAP), and an NO-synthase blocker, NG-nitro-L-arginine methyl ester (LNAME), on transmitter release and processes of exo- and endocytosis of synaptic vesicles in the motornerve ending were studied using electrophysiological and fluorescence techniques. During single stimulation of the motor nerve, SNAP reduced and LNAME did not change the amplitude of the endplate currents and both of the drugs did not affect spontaneous transmitter release. During high-frequency stimulation (20 Hz, 3 min) SNAP increased and LNAME slowed the depression of the amplitudes of endplate potentials (EPPs) compared to the dynamics of EPPs in the control. In experiments using the fluorescent dye FM 1–43, it was shown that the NO donor induced a decrease and LNAME induced an increase in the fluorescence intensity of motor-nerve endings loaded with dye during stimulation at a frequency of 20 Hz for 30 s compared to the control. At the same time, the rate of dye unloading from the terminals that were preloaded with FM 1–43 was higher after preliminary application of SNAP and lower after preliminary application of LNAME. It was suggested that exogenous and endogenous NO in the mouse neuromuscular synapse caused the depression of neurotransmitter release as a result of the suppression of synaptic-vesicle recycling due to a decrease in endocytosis or/and mobilization of synaptic vesicles from a recycling pool to the exocytosis sites.     

A novel function of synapsin II in neurotransmitter release

Although synapsin has been localized to presynaptic structures, its function remains poorly understood. In the present study, we investigated the presynaptic function of synapsin II using a synaptic vesicle recycling process using synapsin-II-overexpressing NG108-15 cells. Western blot analysis with antibodies for synaptic-vesicle-associated protein indicated that the number of synaptic vesicles was approximately doubled in synapsin II transfectants as reported previously. In differentiated synapsin-II-overexpressing and control cells, the application of high potassium induced strong intracellular calcium elevation along neurites and varicosities after differentiation and a weak calcium rise in the cell bodies. The uptake and release of the fluorescent dye FM1-43 revealed that synaptic vesicle recycling in synapsin-II-transfected cells occurred with the same kinetics in the cell body and neuritic varicosities. Furthermore, the area labeled with FM1-43 fluorescence in the synapsin-II-transfected cells was approximately twice as much as in control cells after stimulation, and ATP released after synaptic vesicle fusion with the plasma membrane in synapsin-II-expressing cells was significantly elevated relative to controls. The number of synaptic vesicles paralleled the amount of transmitter released from the cells leading to the conclusion that the number of releasable synaptic vesicles were increased by synapsin II transfection into NG108-15 cells, suggesting that synapsin II may have a role in the regulation of synaptic vesicle number in presynapse-like structures in NG108-15 cells.     

Presynaptic Ultrastructural Plasticity Along CA3→CA1 Axons During Long‐Term Potentiation in Mature Hippocampus

In area CA1 of the mature hippocampus, synaptogenesis occurs within 30 minutes after the induction of long‐term potentiation (LTP); however, by 2 hours many small dendritic spines are lost, and those remaining have larger synapses. Little is known, however, about associated changes in presynaptic vesicles and axonal boutons. Axons in CA1 stratum radiatum were evaluated with 3D reconstructions from serial section electron microscopy at 30 minutes and 2 hours after induction of LTP by theta‐burst stimulation (TBS). The frequency of axonal boutons with a single postsynaptic partner was decreased by 33% at 2 hours, corresponding perfectly to the 33% loss specifically of small dendritic spines (head diameters <0.45 μm). Docked vesicles were reduced at 30 minutes and then returned to control levels by 2 hours following induction of LTP. By 2 hours there were fewer small synaptic vesicles overall in the presynaptic vesicle pool. Clathrin‐mediated endocytosis was used as a marker of local activity, and axonal boutons containing clathrin‐coated pits showed a more pronounced decrease in presynaptic vesicles at both 30 minutes and 2 hours after induction of LTP relative to control values. Putative transport packets, identified as a cluster of less than 10 axonal vesicles occurring between synaptic boutons, were stable at 30 minutes but markedly reduced by 2 hours after the induction of LTP. APV blocked these effects, suggesting that the loss of axonal boutons and presynaptic vesicles was dependent on N‐methyl‐D‐aspartic acid (NMDA) receptor activation during LTP. These findings show that specific presynaptic ultrastructural changes complement postsynaptic ultrastructural plasticity during LTP. J. Comp. Neurol. 521:3898–3912, 2013. © 2013 Wiley Periodicals, Inc.     

Membrane recycling due to low and high rates of nerve stimulation at release sites in the amphibian (Bufo marinus) neuromuscular junction

The activity-dependent labelling of motor nerve terminals with the dye FM1-43 has been used to estimate the relative levels of membrane recycling (due to synaptic vesicle exocytosis and recovery) at release sites in response to 1,200 nerve stimulations delivered at either low (0.5 Hz) or high (30 Hz) frequency. Dye in terminals appears as fluorescent spots distributed along the terminal branches; each spot is thought to be a cluster of labelled vesicles associated with a release site. Relative fluorescence in spots was quantified from images obtained with a confocal microscope. Spot intensities varied widely within branches following labelling at both frequencies, but the distribution was highly skewed towards lower intensities at low frequency stimulation; at high frequency, more spots had stronger fluorescence. Both weak and strongly stained spots were uniformly distributed along the length of terminal branches after low frequency stimulation; however, there was a gradual decline in all spot intensities towards the distal end of branches loaded with dye at high frequency stimulation. Antibody staining for synaptic vesicles was, on average, uniformly distributed along the branches. The increase in number of more strongly FM1-43-labelled spots in terminal branches stimulated at high compared with low frequency suggests that more release sites are active at high rates of nerve stimulation. This "recruitment" of release sites at high frequency stimulation occurs mostly in the proximal half of terminal branches and is not related to the abundance of synaptic vesicles in the terminal.     

L-AP3 blocks rises in intracellular calcium associated with hippocampal CA1 LTP

The involvement of type I metabotropic glutamate receptors in hippocampal CA1 long-term potentiation (LTP) depends on the applied tetanic stimulation protocol. Activation of these receptors may cause an elevation of intracellular calcium via the formation of the second messenger inositol triphosphate (IP3) and subsequent intracellular calcium release. It has been shown that the type I metabotropic receptors antagonist L-2-amino-3-phosphonopropionate (L-AP3) blocks CA1 LTP. Combining dendritic calcium and field potential measurements in CA1 hippocampal area, we found that L-AP3 did not affect single calcium transients but reduced the calcium changes evoked by a single tetanus, preventing the long-lasting calcium enhancements associated with CA1 LTP. These findings suggest that the formation of this type of LTP requires calcium release from IP3-sensitive stores.     

Increase in syntaxin 1B and glutamate release in mossy fibre terminals following induction of LTP in the dentate gyrus: a candidate molecular mechanism underlying transsynaptic plasticity

A growing body of evidence suggests that modulation of certain proteins of the exocytotic machinery is, in part, involved in the biochemical changes that underlie long-term synaptic plasticity. We have previously shown that the induction of long-term potentiation (LTP) at perforant path to dentate granule cell synapses in the rat hippocampus induces changes in the mRNA levels of syntaxin 1B and synapsin I, known to be involved in neurotransmitter release. Immunohistochemical staining suggested that concomitant changes in these proteins occurred at mossy fibre synapses, downstream of those synapses at which LTP was induced, leading us to postulate that such a mechanism might underlie a form of transsynaptic plasticity. Here we have used a specific mossy-fibre synaptosome preparation to quantify levels of proteins and measure, using a chemiluminescent glutamate assay, depolarization-induced glutamate release from these synaptosomes after induction of LTP in the dentate gyrus in vivo. We show that 5 h after the induction of LTP, there is an increase in the protein levels of syntaxin 1B and, although to a lesser extent, the synapsins I and II, associated with an increase in depolarization-induced release of glutamate within these terminals. Increases in both the protein levels and glutamate release were not observed when dentate gyrus LTP was blocked by an NMDA receptor antagonist. From these results we propose a molecular mechanism for the propagation of synaptic plasticity through hippocampal circuits.     

Actin disassembles reversibly during electrically induced recycling of synaptic vesicles in cultured neurons

We have studied depolarization-induced regulation of actin assembly in exocytotically active areas of dissociated chick sympathetic neurons. Active areas were identified with the fluorescent dye FM1-43 which labels synaptic vesicles that recycle in these regions. Exocytosis (electrically stimulated) was monitored in real time through depletion of FM1-43 fluorescence. To study depolarization-induced disassembly of actin in the FM1-43-stained regions, the cells were fixed after different periods of depolarization and stained with rhodamine phalloidin, which binds preferentially to the filamentous form of actin. In active regions, actin disassembles and reassembles during continuous 2 min depolarization. Actin disassembly that occurs after the first 25 s of depolarization was detected by a reduction in rhodamine phalloidin staining and confirmed by correlative electron microscopy. Immunogold staining revealed that actin is abundant throughout resting terminals. In some experiments, actin filaments were stabilized by loading cells with unlabelled phalloidin before stimulating secretion. Stabilizing the filaments does not alter the initial release but strongly reduces the release rate at later stages. These data are consistent with a model in which partial disassembly of actin filaments is necessary for facilitating the transport of vesicles within the terminal and reassembly is necessary for limiting that movement.     

Impaired hippocampal long-term potentiation in microtubule-associated protein 1B-deficient mice

Microtubule-associated protein (MAP)1B-heterozygous (MAP1B+/-) mice are deficient in the expression of MAP1B in the hippocampus, cerebellum, and olfactory cortex. Although MAP1B+/- mice showed half the normal levels of MAP1B protein, they had no measurable amounts of phosphorylated MAP1B. High-frequency theta burst stimulation of Schaffer collateral-CA1 axons in hippocampal slices from MAP1B+/- mice elicited long-term potentiation (LTP) that decayed rapidly to baseline, in contrast to the non-decremental LTP exhibited by age-matched wild-type slices. A separate group of MAP1B+/- and wild-type slices was examined for a longer time course of 3 hr post-tetanus in response to multiple high-frequency stimulus trains that induced saturated LTP. MAP1B+/- slices showed marked reductions in both immediate post-tetanic potentiation and LTP that decayed much more rapidly than that in wild-type slices. The induction of LTP was associated with a rapid dephosphorylation of MAP1B within 5-15 min post-tetanus, suggesting that the normal expression of MAP1B and conversion to a dephosphorylated state may be a cellular mediator of cytoskeletal alterations necessary for long-term activity-dependent synaptic plasticity.     

Long-lasting modulation of the induction of LTD and LTP in rat hippocampal CA1 by behavioural stress and environmental enrichment

Behavioural experience (e.g. chronic stress, environmental enrichment) can have long-lasting effects on cognitive functions. Because activity-dependent persistent changes in synaptic strength are believed to mediate memory processes in brain areas such as hippocampus, we tested whether behaviour has also long-lasting effects on synaptic plasticity by examining the induction of long-term potentiation (LTP) and long-term depression (LTD) in slices of hippocampal CA1 obtained from rats either 7-9 months after social defeat (behavioural stress) or 3-5 weeks after 5-week exposure to environmental enrichment. Compared with age-matched controls, defeated rats showed markedly reduced LTP. LTP was even completely impaired but LTD was enhanced in defeated and, subsequently, individually housed (during the 7-9-month period after defeat) rats. However, increasing stimulus intensity during 100-Hz stimulation resulted in significant LTP. This suggests that the threshold for LTP induction is still raised and that for LTD lowered several months after a short stressful experience. Both LTD and LTP were enhanced in environmentally enriched rats, 3-5 weeks after enrichment, as compared with age-matched controls. Because enrichment reduced paired-pulse facilitation, an increase in presynaptic release, facilitating both LTD and LTP induction, might contribute to enhanced synaptic changes. Consistently, enrichment reduced the number of 100-Hz stimuli required for inducing LTP. But enrichment may also actually enhance the range of synaptic modification. Repeated LTP and LTD induction produced larger synaptic changes in enriched than in control rats. These data reveal that exposure to very different behavioural experiences can produce long-lasting effects on the susceptibility to synaptic plasticity, involving pre- and postsynaptic processes.     

A readily releasable pool of single inhibitory boutons in culture

The number of presynaptic vesicles that are immediately available for release, the readily releasable pool (RRP), is a strong determinant of synaptic strength and plasticity. The properties of the RRP in individual GABAergic synapses were examined in superior colliculus cultures. The RRP was depleted by high frequency trains and cumulative evoked IPSC amplitudes (CA) were calculated. The amplitude of monoquantal responses (q) was determined on the basis of mIPSC histograms. On average, the RRP, defined as CA/q, comprised about 10 vesicles. About 60% of the RRP could be released by a single stimulus. After depletion, the RRP was replenished with a time constant of about 14 s. These data provide information for further studies on the capacity of individual inhibitory synapses to modulate sensory information transfer.