Vesicle pool partitioning influences presynaptic diversity and weighting in rat hippocampal synapses

Hippocampal synapses display a range of release probabilities. This is partially the result of scaling of release probability with the total number of releasable vesicles at each synapse. We have compared synaptic release and vesicle pool sizes across a large number of hippocampal synapses using FM 1-43 and confocal fluorescence microscopy. We found that the relationship between the number of recycling vesicles at a synapse and its release probability is dependent on firing frequency. During firing at 10 Hz, the release probability of each synapse is closely related to the number of recycling vesicles that it contains. In contrast, during firing at 1 Hz, different synapses turn over their recycling vesicle pools at different rates leading to an indirect relationship between recycling vesicle pool size and release probability. Hence two synapses may release vesicles at markedly different rates during low frequency firing, even if they contain similar numbers of vesicles. Both further kinetic analyses and manipulation of the number of vesicles in the readily releasable pool using phorbol ester treatment suggested that this imprecise scaling observed during firing at 1 Hz resulted from synapse-to-synapse differences in the proportion of recycling vesicles partitioned into the readily releasable pool. Hence differential partitioning between vesicle pools affects presynaptic weighting in a frequency-dependent manner. Since hippocampal single unit firing rates shift between 1 Hz and 10 Hz regimes with behavioural state, differential partitioning may be a mechanism for encoding information in hippocampal circuits.     

Diazepam attenuates the post-traumatic hyperactivity of excitatory synapses in rat hippocampal CA1 neurons

The effect of diazepam, a benzodiazepine derivative, on the post-traumatic hyperactivity of excitatory synaptic transmission was examined in rat hippocampal CA1 area. Optical recordings showed that the activity of hippocampal neurons was enhanced in rats treated with fluid percussion injury (FPI) as compared with that of sham-operated rats. The optical response was characterized by fast and slow components. FPI did not affect the fast component that reflects presynaptic action potentials, but enhanced the slow component that reflects excitatory synaptic responses. Intracellular recordings showed that the amplitude and duration of the excitatory postsynaptic potential (EPSP) were increased after FPI. However, FPI did not affect the resting membrane potential and action potentials of hippocampal neurons. Intraperitoneal (i.p.) administration of diazepam (30 and 90 min after FPI) attenuated the post-traumatic hyperactivity of the slow optical response. The slope of input-to-output relation of excitatory synapses was decreased by acute administration of diazepam to FPI rats, but not by delayed administration of diazepam (4 and 5 h after FPI). The fast optical responses were not affected by either FPI or i.p. administration of diazepam. These results suggest that administration of diazepam at early post-traumatic period prevents the FPI-induced delayed enhancement of excitatory synaptic transmission in rat hippocampal CA1 neurons.     

Realistic modelling of receptor activation in hippocampal excitatory synapses: analysis of multivesicular release, release location, temperature and synaptic cross-talk

Chemically mediated synaptic transmission results from fusion of synaptic vesicles with the presynaptic plasma membrane, subsequent release of the vesicular content into the cleft and binding to postsynaptic receptors. Previous modelling studies of excitatory neurotransmitter glutamate were based on simplified geometries failing to account for the biologically realistic synaptic environment, in particular, the presence of astrocytes, the geometry of extracellular space, and the neurotransmitter uptake mechanism. Using 3-dimensional reconstructions of hippocampal glutamatergic synapses including the surrounding astrocytic processes we have developed a biologically realistic model to analyse receptor activation in different conditions. We used the finite element method to simulate glutamate release, analyse glutamate diffusion following single and multiple vesicle release and binding at the postsynaptic site to AMPA and NMDA receptors. We demonstrate that: (1) the transmitter diffusion is highly temperature-sensitive; (2) release conditions and geometry more specifically affect AMPARs than NMDARs; (3) the sensitivities of AMPARs and NMDARs to simultaneous vesicular release are different; (4) in the case of multivesicle neurotransmitter release with variable delays, the binding of glutamate to AMPARs is additive up to 1 ms after the release, then becomes independent, but to NMDARs the binding is additive up to 33 ms; (5) the number of AMPARs varies more than the number of NMDRs in response to the input firing patterns; (6) the presence of astrocytes effectively blocks synaptic cross-talk; and (7) synaptic cross-talk, mediated by NMDARs but not AMPARs, is only possible after quasi-simultaneous multivesicular release at physiological temperature (35°C) without intervening astrocytes, but not at 25°C. Our simulations demonstrate the importance of temperature and ultrastructural synaptic environment in synaptic transmission and synaptic cross-talk.     

Clathrin-mediated endocytosis: the physiological mechanism of vesicle retrieval at hippocampal synapses

The maintenance of synaptic transmission requires that vesicles are recycled after releasing neurotransmitter. Several modes of retrieval have been proposed to operate at small synaptic terminals of central neurons, but the relative importance of these has been controversial. It is established that synaptic vesicles can collapse on fusion and the machinery for retrieving this membrane by clathrin-mediated endocytosis (CME) is enriched in the presynaptic terminal. But it has also been suggested that the majority of vesicles released by physiological stimulation are recycled by a second, faster mechanism called 'kiss-and-run', which operates in 1 s or less to retrieve a vesicle before it has collapsed. The most recent evidence argues against the occurrence of 'kiss-and-run' in hippocampal synapses. First, an improved fluorescent reporter of exocytosis (sypHy), indicates that only a slow mode of endocytosis  (τ= 15 s)  operates when vesicle fusion is triggered by a single nerve impulse or short burst. Second, this retrieval mechanism is blocked by overexpressing the C-terminal fragment of AP180 or by knockdown of clathrin using RNAi. Third, vesicle fusion is associated with the movement of clathrin and vesicle proteins out of the synapse into the neighbouring axon. These observations indicate that clathrin-mediated endocytosis is the major, if not exclusive, mechanism of retrieval in small hippocampal synapses.     

Synaptic vesicles recycling spontaneously and during activity belong to the same vesicle pool

Recently, it has been claimed that vesicles recycling spontaneously and during activity belong to different pools. Here we simultaneously measured, using spectrally separable styryl dyes, the release kinetics of vesicles recycled spontaneously or upon stimulation and the effects of the v-ATPase blocker folimycin on the frequency of miniature postsynaptic currents in rat hippocampal neurons. Our results provide evidence as to the identities of the vesicle pools recycling at rest and during stimulation.     

Matrix metalloproteinase-7 modulates synaptic vesicle recycling and induces atrophy of neuronal synapses

Matrix metalloproteinase-7 (MMP-7) belongs to a family of zinc dependent endopeptidases that are expressed in a variety of tissues including the brain. MMPs are known to be potent mediators of pericellular proteolysis and likely mediators of dynamic remodelling of neuronal connections. While an association between proteases and the neuronal synapse is emerging, a full understanding of this relationship is lacking. Here, we show that MMP-7 alters the structure and function of presynaptic terminals without affecting neuronal survival. Bath application of recombinant MMP-7 to cultured rat neurons induced long-lasting inhibition of vesicular recycling as measured by synaptotagmin 1 antibody uptake assays and FM4-64 optical imaging. MMP-7 application resulted in reduced abundance of vesicular and active zone proteins locally within synaptic terminals although their general levels remained unaltered. Finally, chronic application of the protease resulted in synaptic atrophy, including smaller terminals and fewer synaptic vesicles, as determined by electron microscopy. Together these results suggest that MMP-7 is a potent modulator of synaptic vesicle recycling and synaptic ultrastructure and that elevated levels of the enzyme, as may occur with brain inflammation, may adversely influence neurotransmission.     

Discharge of the readily releasable pool with action potentials at hippocampal synapses

A readily releasable pool (RRP) of synaptic vesicles has been identified at hippocampal synapses with application of hypertonic solution. RRP size correlates with important properties of synaptic function such as release probability. However, a discrepancy in RRP size has been reported depending on the method used to evoke synaptic release. This study was undertaken to determine quantitative relationships between the RRP defined with hypertonic solution and that released with trains of action potentials. We find that asynchronous release at cell culture synapses contributes significantly to the discharge of the RRP with trains of action potentials and that RRP size is the same when elicited by either nerve stimuli or hypertonic challenge.     

Local structural balance and functional interaction of excitatory and inhibitory synapses in hippocampal dendrites

Theoretical and experimental studies on the computation of neural networks suggest that neural computation results from a dynamic interplay of excitatory and inhibitory (E/I) synaptic inputs. Precisely how E/I synapses are organized structurally and functionally to facilitate meaningful interaction remains elusive. Here we show that E/I synapses are regulated across dendritic trees to maintain a constant ratio of inputs in cultured rat hippocampal neurons. This structural arrangement is accompanied by an E/I functional balance maintained by a 'push-pull' feedback regulatory mechanism that is capable of adjusting E/I efficacies in a coordinated fashion. We also found that during activity, inhibitory synapses can determine the impact of adjacent excitatory synapses only if they are colocalized on the same dendritic branch and are activated simultaneously. These fundamental relationships among E/I synapses provide organizational principles relevant to deciphering the structural and functional basis for neural computation within dendritic branches.     

Total number and distribution of inhibitory and excitatory synapses on hippocampal CA1 pyramidal cells

The integrative properties of neurons depend strongly on the number, proportions and distribution of excitatory and inhibitory synaptic inputs they receive. In this study the three-dimensional geometry of dendritic trees and the density of symmetrical and asymmetrical synapses on different cellular compartments of rat hippocampal CA1 area pyramidal cells was measured to calculate the total number and distribution of excitatory and inhibitory inputs on a single cell.A single pyramidal cell has approximately 12,000 microm dendrites and receives around 30,000 excitatory and 1700 inhibitory inputs, of which 40 % are concentrated in the perisomatic region and 20 % on dendrites in the stratum lacunosum-moleculare. The pre- and post-synaptic features suggest that CA1 pyramidal cell dendrites are heterogeneous. Strata radiatum and oriens dendrites are similar and differ from stratum lacunosum-moleculare dendrites. Proximal apical and basal strata radiatum and oriens dendrites are spine-free or sparsely spiny. Distal strata radiatum and oriens dendrites (forming 68.5 % of the pyramidal cells' dendritic tree) are densely spiny; their excitatory inputs terminate exclusively on dendritic spines, while inhibitory inputs target only dendritic shafts. The proportion of inhibitory inputs on distal spiny strata radiatum and oriens dendrites is low ( approximately 3 %). In contrast, proximal dendritic segments receive mostly (70-100 %) inhibitory inputs. Only inhibitory inputs innervate the somata (77-103 per cell) and axon initial segments. Dendrites in the stratum lacunosum-moleculare possess moderate to small amounts of spines. Excitatory synapses on stratum lacunosum-moleculare dendrites are larger than the synapses in other layers, are frequently perforated ( approximately 40 %) and can be located on dendritic shafts. Inhibitory inputs, whose percentage is relatively high ( approximately 14-17 %), also terminate on dendritic spines.Our results indicate that: (i) the highly convergent excitation arriving onto the distal dendrites of pyramidal cells is primarily controlled by proximally located inhibition; (ii) the organization of excitatory and inhibitory inputs in layers receiving Schaffer collateral input (radiatum/oriens) versus perforant path input (lacunosum-moleculare) is significantly different.     

Kinetic isolation of a slowly recovering component of short-term depression during exhaustive use at excitatory hippocampal synapses

This study examines the kinetics of the longest lasting form of short-term depression at excitatory hippocampal synapses. After initial depletion of the readily releasable pool (RRP), continued 20-Hz stimulation was found to be fast enough to maximally drive presynaptic neurotransmitter exocytosis; maximal is defined here as the rate needed to maintain the RRP in a nearly empty steady state. Induction of depression proceeded in two distinct phases. The first was caused by RRP depletion, whereas the second is shown to reflect the progressive reduction of the overall rate at which new vesicles are supplied to the RRP and is termed "supply-rate depression." Supply-rate depression is identified further with the emergence, during heavy use, of a rate-limiting vesicle trafficking step that slows the timing of RRP replenishment by switching from a fast (tau congruent with 7 s) to a slow (tau congruent with 1 min) vesicle supply mechanism. Both mechanisms apparently follow first-order kinetics. After the induction of the maximum amount of depression, individual synapses were able to output only <1 quantum of neurotransmitter per synapse per second, matching previous predictions based on cell biological measurements of synaptic vesicle cycling. Surprisingly, the onset of supply-rate depression occurred with a marked delay, not having a detectable impact on synaptic function until after several seconds of continuous use. The delayed onset is not consistent with traditional vesicle trafficking models, but may be important for limiting the impact of supply-rate depression to pathological episodes and might function as a native antiepilepsy device.     

Analysis of evoked and spontaneous quantal release at high pressure in crustacean excitatory synapses

The cellular mechanisms underlying the effect of high pressure on synaptic transmission were studied in the opener muscle of the lobster walking leg. Excitatory postsynaptic currents (EPSCs) were recorded using a loose macropatch-clamp technique at normal pressure and 3.5, 6.9 MPa helium pressure. Responses of the single excitatory axon could be grouped into two types: low-yield (L) synapses exhibiting small EPSCs with a considerable number of failures, and high-yield (H) synapses having larger EPSCs with very few failures. High pressure reduced the average EPSC amplitude in all synapses and shifted their amplitude histograms to the left by decreasing the quantal content (m) without changing their quantum current (q). A binomial distribution fit of EPSC amplitudes revealed that high pressure greatly decreased n, the number of available active zones, but the effect on p, the probability of release for each zone, was not consistent. Many of the spontaneous miniature EPSCs (mEPSCs), observed only in L-type synapses, were giant (size=2–5 q). High pressure increased the frequency of the giant mEPSCs but had little effect on their amplitude histogram. High pressure depressed evoked synaptic transmission by modulating the presynaptic quantal release parameters, but concomitantly enhanced spontaneous quantal release by an unknown mechanism.     

Large dense-core vesicle exocytosis and membrane recycling in the mossy fibre synapses of the rabbit hippocampus during epileptiform seizures

Summary The ultrastructure of the hippocampal mossy fibre layer was studied in ultrathin sections and freeze-fracture preparations of rabbits under deep Nembutal anaesthesia, after recovery from ether anaesthesia, and 40 min after a single injection of methoxypyridoxine, that is, during the second generalized seizure discharge. The giant mossy fibre boutons contain two types of vesicles: evenly distributed, small round clear vesicles (50 nm) and a few scattered large dense-core vesicles (100 nm). In rare instances fusion of dense-core vesicles with the presynaptic membrane was observed. No differences in the morphology of the mossy fibre synapses were found between anaesthetized and unanaesthetized animals. During epileptiform seizures, however, the size and shape of clear and dense-core vesicles varied greatly. The active synaptic zones were covered with large, core-containing omega profiles or bumps and indentations. Only dense-core vesicles seem to undergo exocytosis. A fusion of clear vesicles with the presynaptic membrane was not observed.Various explanations for the fact that only dense-core vesicles seem to undergo exocytosis are discussed. The hypothesis is put forward that in the mossy fibre bouton two morphologically and functionally distinct populations of synaptic vesicles exist and that only one of them undergoes visible irreversible exocytosis, whereas the majority, that is, the small vesicles discharge their transmitter by reversible fusion.After MP injection features of membrane retrieval were also prominent. Frequently, at the borders of the active synaptic zones coated membrane convolutes of both pre- and postsynaptic membranes had invaded the terminals as well as the postsynaptic spine. Thus, in contrast to electrical stimulation, the self-sustained seizures allow energy-expensive processes such as extensive membrane internalization to take place during the interictal pauses.     

Aerobic exercise improves hippocampal function and increases BDNF in the serum of young adult males

Physical activity has been reported to improve cognitive function in humans and rodents, possibly via a brain-derived neurotrophic factor (BDNF)-regulated mechanism. In this study of human subjects, we have assessed the effects of acute and chronic exercise on performance of a face-name matching task, which recruits the hippocampus and associated structures of the medial temporal lobe, and the Stroop word-colour task, which does not, and have assessed circulating concentrations of BDNF and IGF-1 in parallel. The results show that a short period of high-intensity cycling results in enhancements in performance of the face-name matching, but not the Stroop, task. These changes in cognitive function were paralleled by increased concentration of BDNF, but not IGF-1, in the serum of exercising subjects. 3 weeks of cycling training had no effect on cardiovascular fitness, as assessed by VO₂ scores, cognitive function, or serum BDNF concentration. Increases in fitness, cognitive function and serum BDNF response to acute exercise were observed following 5 weeks of aerobic training. These data indicate that both acute and chronic exercise improve medial temporal lobe function concomitant with increased concentrations of BDNF in the serum, suggesting a possible functional role for this neurotrophic factor in exercise-induced cognitive enhancement in humans.