Actin study of the synapses of surviving hippocampal slices during long-term potentiation

A study was made of the synaptic actin ultrastructural localization in the hippocampal slices at long-lasting potentiation of area CA, using myosin subfragment-1 labeling. A specific qualitative ultrastructural sign of the potentiated hippocampal synapses was revealed for the first time - the formation in spines of rodlike bundles of actin filaments resembling the cilia. They penetrate the spine stalks to pass through the spine core towards the postsynaptic densities of active zones. The thinner bridges link the filament bundles with the actin cytoskeleton meshwork, with spine apparatus cisterns and with postsynaptic membranes of the active zones. Besides the increasing density of the presynaptic actin meshwork was shown. The changes in the actin cytoskeleton being taken into consideration, its contractile properties account for some morphofunctional features of the potentiated synapses known before and predict previously unknown ones.     

Short term synaptic depression imposes a frequency dependent filter on synaptic information transfer

Depletion of synaptic neurotransmitter vesicles induces a form of short term depression in synapses throughout the nervous system. This plasticity affects how synapses filter presynaptic spike trains. The filtering properties of short term depression are often studied using a deterministic synapse model that predicts the mean synaptic response to a presynaptic spike train, but ignores variability introduced by the probabilistic nature of vesicle release and stochasticity in synaptic recovery time. We show that this additional variability has important consequences for the synaptic filtering of presynaptic information. In particular, a synapse model with stochastic vesicle dynamics suppresses information encoded at lower frequencies more than information encoded at higher frequencies, while a model that ignores this stochasticity transfers information encoded at any frequency equally well. This distinction between the two models persists even when large numbers of synaptic contacts are considered. Our study provides strong evidence that the stochastic nature neurotransmitter vesicle dynamics must be considered when analyzing the information flow across a synapse.     

Proteomics, ultrastructure, and physiology of hippocampal synapses in a fragile X syndrome mouse model reveal presynaptic phenotype

Fragile X syndrome (FXS), the most common form of hereditary mental retardation, is caused by a loss-of-function mutation of the Fmr1 gene, which encodes fragile X mental retardation protein (FMRP). FMRP affects dendritic protein synthesis, thereby causing synaptic abnormalities. Here, we used a quantitative proteomics approach in an FXS mouse model to reveal changes in levels of hippocampal synapse proteins. Sixteen independent pools of Fmr1 knock-out mice and wild type mice were analyzed using two sets of 8-plex iTRAQ experiments. Of 205 proteins quantified with at least three distinct peptides in both iTRAQ series, the abundance of 23 proteins differed between Fmr1 knock-out and wild type synapses with a false discovery rate (q-value) <5%. Significant differences were confirmed by quantitative immunoblotting. A group of proteins that are known to be involved in cell differentiation and neurite outgrowth was regulated; they included Basp1 and Gap43, known PKC substrates, and Cend1. Basp1 and Gap43 are predominantly expressed in growth cones and presynaptic terminals. In line with this, ultrastructural analysis in developing hippocampal FXS synapses revealed smaller active zones with corresponding postsynaptic densities and smaller pools of clustered vesicles, indicative of immature presynaptic maturation. A second group of proteins involved in synaptic vesicle release was up-regulated in the FXS mouse model. In accordance, paired-pulse and short-term facilitation were significantly affected in these hippocampal synapses. Together, the altered regulation of presynaptically expressed proteins, immature synaptic ultrastructure, and compromised short-term plasticity points to presynaptic changes underlying glutamatergic transmission in FXS at this stage of development.     

Synaptogenesis of hippocampal neurons in primary cell culture

Hippocampal neurons in dissociated cell culture are one of the most extensively used model systems in the field of molecular and cellular neurobiology. Only limited data are however available on the normal time frame of synaptogenesis, synapse number and ultrastructure of excitatory synapses during early development in culture. Therefore, we analyzed the synaptic ultrastructure and morphology and the localization of presynaptic (Bassoon) and postsynaptic (ProSAP1/Shank2) marker proteins in cultures established from rat embryos at embryonic day 19, after 3, 7, 10, 14, and 21 days in culture. First excitatory synapses were identified at day 7 with a clearly defined postsynaptic density and presynaptically localized synaptic vesicles. Mature synapses on dendritic spines were seen from day 10 onward, and the number of synapses steeply increased in the third week. Fenestrated or multiple synapses were found after 14 or 21 days, respectively. So-called dense-core vesicles, responsible for the transport of proteins to the active zone of the presynaptic specialization, were seen on cultivation day 3 and 7 and could be detected in axons and especially in the presynaptic subcompartments. The expression and localization of the presynaptic protein Bassoon and of the postsynaptic molecule ProSAP1/Shank2 was found to correlate nicely with the ultrastructural results. This regular pattern of development and maturation of excitatory synapses in hippocampal culture starting from day 7 in culture should ease the comparison of synapse number and morphology of synaptic contacts in this widely used model system.     

Synaptic development: insights from Drosophila

In Drosophila, the larval neuromuscular junction is particularly tractable for studying how synapses develop and function. In contrast to vertebrate central synapses, each presynaptic motor neuron and postsynaptic muscle cell is unique and identifiable, and the wiring circuit is invariant. Thus, the full power of Drosophila genetics can be brought to bear on a single, reproducibly identifiable, synaptic terminal. Each individual neuromuscular junction encompasses hundreds of synaptic neurotransmitter release sites housed in a chain of synaptic boutons. Recent advances have increased our understanding of the mechanisms that shape the development of both individual synapses--that is, the transmitter release sites including active zones and their apposed glutamate receptor clusters--and the whole synaptic terminal that connects a pre- and post-synaptic cell.     

Assembling the presynaptic active zone: a characterization of an active one precursor vesicle

The active zone is a specialized region of the presynaptic plasma membrane where synaptic vesicles dock and fuse. In this study, we have investigated the cellular mechanism underlying the transport and recruitment of the active zone protein Piccolo into nascent synapses. Our results show that Piccolo is transported to nascent synapses on an approximately 80 nm dense core granulated vesicle together with other constituents of the active zone, including Bassoon, Syntaxin, SNAP-25, and N-cadherin, as well as chromogranin B. Components of synaptic vesicles, such as VAMP 2/synaptobrevin II, synaptophysin, synaptotagmin, or proteins of the perisynaptic plasma membrane such as GABA transporter 1 (GAT1), were not present. These studies demonstrate that the presynaptic active zone is formed in part by the fusion of an active zone precursor vesicle with the presynaptic plasma membrane.     

Postsynaptic Y654 dephosphorylation of β‐catenin modulates presynaptic vesicle turnover through increased n‐cadherin‐mediated transsynaptic signaling

Synaptic adhesion molecules, which coordinately control structural and functional changes at both sides of synapses, are important for synaptogenesis and synaptic plasticity. Because they physically form homophilic or heterophilic adhesions across synaptic junctions, these molecules can initiate transsynaptic communication in both anterograde and retrograde directions. Using optical imaging approaches, we investigated whether an increase in postsynaptic N‐cadherin could correspondingly alter the function of connected presynaptic terminals. Postsynaptic expression of β‐catenin Y654F, a phosphorylation‐defective form with enhanced binding to N‐cadherin, is sufficient to increase postsynaptic surface levels of N‐cadherin and consequently promote presynaptic reorganizations. Such reorganizations include increases in the densities of the synaptic vesicle protein, Synaptotagmin 1 and the active zone scaffold protein, Bassoon, the number of active boutons and the size of the total recycling vesicle pool. In contrast, synaptic vesicle turnover is significantly impaired, preventing the exchange of synaptic vesicles with adjacent boutons. Together, N‐cadherin‐mediated retrograde signaling, governed by phosphoregulation of postsynaptic β‐catenin Y654, coordinately modulates presynaptic vesicle dynamics to enhance synaptic communication in mature neurons. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 61–74, 2017     

Somato-dendritic synapses in the nucleus reticularis thalami of the rat

In the reticular nucleus of the rat thalamus, about 30% of the synapses are brought about by the perikarya of parvalbumin-immunopositive neurons, which establish somato-dendritic synapses with large dendrites of nerve cells of specific thalamic nuclei. Although the parvalbumin-immunopositive presynaptic structures bear resemblance to goblet-like or calyciform axonal endings, electron microscopic immunocytochemistry and in situ hybridization revealed that these structures are parts of the perikaryal cytoplasm studded with synaptic vesicles. In about 15% of the somato-dendritic synapses, axons are seen to be in synaptic contact with the parvalbumin-immunoreactive perikaryon. Double immunohistochemical staining revealed that the parvalbumin immunoreactive presynaptic perikarya and dendrites contained GABA. It is assumed that the peculiar somato-dendritic synaptic complexes subserve the goal of filtration of impulses arriving at the reticular nucleus from various thalamic nuclei, thus processing them for further sampling.     

Local presynaptic activity gates homeostatic changes in presynaptic function driven by dendritic BDNF synthesis

Homeostatic synaptic plasticity is important for maintaining stability of neuronal function, but heterogeneous expression mechanisms suggest that distinct facets of neuronal activity may shape the manner in which compensatory synaptic changes are implemented. Here, we demonstrate that local presynaptic activity gates a retrograde form of homeostatic plasticity induced by blockade of AMPA receptors (AMPARs) in cultured hippocampal neurons. We show that AMPAR blockade produces rapid (<3 hr) protein synthesis-dependent increases in both presynaptic and postsynaptic function and that the induction of presynaptic, but not postsynaptic, changes requires coincident local activity in presynaptic terminals. This "state-dependent" modulation of presynaptic function requires postsynaptic release of brain-derived neurotrophic factor (BDNF) as a retrograde messenger, which is locally synthesized in dendrites in response to AMPAR blockade. Taken together, our results reveal a local crosstalk between active presynaptic terminals and postsynaptic signaling that dictates the manner by which homeostatic plasticity is implemented at synapses.     

Communicating synapses: types and functional interpretation. Exceptions to Cajal's neuron theory.

The neurons of the dorsal periaqueductal nucleus of the mesencephalon and their synaptic contacts were observed under a transmission electron microscope. We found various types of synapses which constituted an exception to Cajal's neuron theory (law of neuron independence). Some of these synapses had an open communicating or continuity 'passage' between the presynaptic bouton of a neuron (first neuron) and the postsynaptic portion of another neuron (second neuron). The 'communicating' passage (located in the synaptosome) is formed by the continuity of the presynaptic and postsynaptic membrane, and its limits or rims are the reflexion points of the membranes. When only two neurons intervene they could be termed 'simple communicating synapses'. We found three types: I = communicating axosomatic synapses; II = communicating axodendritic synapses, and III = communicating axoaxonic synapses'. When three neurons intervene in the synaptic contact, they could be termed 'complex communicating synapses'. In these, the first and second neurons form a normal synapse, but the lateral portion of the presynaptic bouton of the first neuron also enters into contact with a third neuron, with which it establishes an open communicating or continuity passage. The points of these passages are collateral to the synapse, and may be in the presynaptic or pre-postsynaptic portions simultaneously, communicating collaterally with the third neuron. We found a further three types: IV = complex communicating axosomatic and dendritic synapses; V = complex communicating axoaxonic and somatic synapses, and VI = complex communicating axodendritic and double-somatic synapses. It is suggested that communicating synapses may constitute an exception to Cajal's neuron theory, representing functional states for the acceleration, retardation or modulation of the synaptic function. The neurotransmitters would pass en masse through the communicating passage and the depolarization wave would pass through the rims without being retarded. In the simple communicating synapses, their action would be intensifying. In the complex communicating synapses, their action would be modulating or retarding, since the collateral communicating passage would function as an 'escape valve' through which part of the impulse reaching the presynaptic bouton would escape.     

Pro-BDNF–induced synaptic depression and retraction at developing neuromuscular synapses

Postsynaptic cells generate positive and negative signals that retrogradely modulate presynaptic function. At developing neuromuscular synapses, prolonged stimulation of muscle cells induces sustained synaptic depression. We provide evidence that pro–brain-derived neurotrophic factor (BDNF) is a negative retrograde signal that can be converted into a positive signal by metalloproteases at the synaptic junctions. Application of pro-BDNF induces a dramatic decrease in synaptic efficacy followed by a retraction of presynaptic terminals, and these effects are mediated by presynaptic pan-neurotrophin receptor p75 (p75^NTR), the pro-BDNF receptor. A brief stimulation of myocytes expressing cleavable or uncleavable pro-BDNF elicits synaptic potentiation or depression, respectively. Extracellular application of metalloprotease inhibitors, which inhibits the cleavage of endogenous pro-BDNF, facilitates the muscle stimulation–induced synaptic depression. Inhibition of presynaptic p75^NTR or postsynaptic BDNF expression also blocks the activity-dependent synaptic depression and retraction. These results support a model in which postsynaptic secretion of a single molecule, pro-BDNF, may stabilize or eliminate presynaptic terminals depending on its proteolytic conversion at the synapses.     

Hair cell ribbon synapses

Hearing and balance rely on the faithful synaptic coding of mechanical input by the auditory and vestibular hair cells of the inner ear. Mechanical deflection of their stereocilia causes the opening of mechanosensitive channels, resulting in hair cell depolarization, which controls the release of glutamate at ribbon-type synapses. Hair cells have a compact shape with strong polarity. Mechanoelectrical transduction and active membrane turnover associated with stereociliar renewal dominate the apical compartment. Transmitter release occurs at several active zones along the basolateral membrane. The astonishing capability of the hair cell ribbon synapse for temporally precise and reliable sensory coding has been the subject of intense investigation over the past few years. This research has been facilitated by the excellent experimental accessibility of the hair cell. For the same reason, the hair cell serves as an important model for studying presynaptic Ca(2+) signaling and stimulus-secretion coupling. In addition to common principles, hair cell synapses differ in their anatomical and functional properties among species, among the auditory and vestibular organs, and among hair cell positions within the organ. Here, we briefly review synaptic morphology and connectivity and then focus on stimulus-secretion coupling at hair cell synapses.     

Scribble Interacts with β-Catenin to Localize Synaptic Vesicles to Synapses

An understanding of how synaptic vesicles are recruited to and maintained at presynaptic compartments is required to discern the molecular mechanisms underlying presynaptic assembly and plasticity. We have previously demonstrated that cadherin–β-catenin complexes cluster synaptic vesicles at presynaptic sites. Here we show that scribble interacts with the cadherin–β-catenin complex to coordinate vesicle localization. Scribble and β-catenin are colocalized at synapses and can be coimmunoprecipitated from neuronal lysates, indicating an interaction between scribble and β-catenin at the synapse. Using an RNA interference approach, we demonstrate that scribble is important for the clustering of synaptic vesicles at synapses. Indeed, in scribble knockdown cells, there is a diffuse distribution of synaptic vesicles along the axon, and a deficit in vesicle recycling. Despite this, synapse number and the distribution of the presynaptic active zone protein, bassoon, remain unchanged. These effects largely phenocopy those observed after ablation of β-catenin. In addition, we show that loss of β-catenin disrupts scribble localization in primary neurons but that the localization of β-catenin is not dependent on scribble. Our data supports a model by which scribble functions downstream of β-catenin to cluster synaptic vesicles at developing synapses.     

Transsynaptic channelosomes

Recent findings demonstrate that synaptic channels are directly involved in the formation and maintenance of synapses by interacting with synapse organizers. The synaptic channels on the pre- and postsynaptic membranes possess non-conducting roles in addition to their functional roles as ion-conducting channels required for synaptic transmission. For example, presynaptic voltage-dependent calcium channels link the target-derived synapse organizer laminin β2 to cytomatrix of the active zone and function as scaffolding proteins to organize the presynaptic active zones. Furthermore, postsynaptic δ2-type glutamate receptors organize the synapses by forming transsynaptic protein complexes with presynaptic neurexins through synapse organizer cerebellin 1 precursor proteins. Interestingly, the synaptic clustering of AMPA receptors is regulated by neuronal activity-regulated pentraxins, while postsynaptic differentiation is induced by the interaction of postsynaptic calcium channels and thrombospondins. This review will focus on the non-conducting functions of ion-channels that contribute to the synapse formation in concert with synapse organizers and active-zone-specific proteins.     

Presynaptic kainate receptors are localized close to release sites in rat hippocampal synapses

The subsynaptic distribution of kainate receptors is still a matter of much debate given its importance to understand the way they influence neuronal communication. Here, we show that, in synapses of the rat hippocampus, presynaptic kainate receptors are localized within the presynaptic active zone close to neurotransmitter release sites. The activation of these receptors with low concentrations of agonists induces the release of [(3)H]glutamate in the absence of a depolarizing stimulus. Furthermore, this modulation of [(3)H]glutamate release by kainate is more efficient when compared with a KCl-evoked depolarization that causes a more than two-fold increase in the intra-terminal calcium concentration but no apparent release of [(3)H]glutamate, suggesting a direct receptor-mediated process. Using a selective synaptic fractionation technique that allows for a highly efficient separation of presynaptic, postsynaptic and non-synaptic proteins we confirmed that, presynaptically, kainate receptors are mainly localized within the active zone of hippocampal synapses where they are expected to be in a privileged position to modulate synaptic phenomena.     

Transsynaptic channelosomes: non-conducting roles of ion channels in synapse formation

Recent findings demonstrate that synaptic channels are directly involved in the formation and maintenance of synapses by interacting with synapse organizers. The synaptic channels on the pre- and postsynaptic membranes possess non-conducting roles in addition to their functional roles as ion-conducting channels required for synaptic transmission. For example, presynaptic voltage-dependent calcium channels link the target-derived synapse organizer laminin β2 to cytomatrix of the active zone and function as scaffolding proteins to organize the presynaptic active zones. Furthermore, postsynaptic δ2-type glutamate receptors organize the synapses by forming transsynaptic protein complexes with presynaptic neurexins through synapse organizer cerebellin 1 precursor proteins. Interestingly, the synaptic clustering of AMPA receptors is regulated by neuronal activity-regulated pentraxins, while postsynaptic differentiation is induced by the interaction of postsynaptic calcium channels and thrombospondins. This review will focus on the non-conducting functions of ion-channels that contribute to the synapse formation in concert with synapse organizers and active-zone-specific proteins.     

Synaptic potential in the motor giant axon of the crayfish

Some electrical properties of the synapses between central giant axons (presynaptic) and the motor giant axon (postsynaptic) of the crayfish abdominal nerve cord have been investigated. Postsynaptic potential change in response to presynaptic volleys contains two components: a spike potential and a synaptic potential of very long time course. Amplitude of the synaptic potential is graded according to the number of active presynaptic axons. Conductance increase in the synaptic membrane endures over most of the period of potential change, and it is this rather than the "electrical time constant" of the membrane that in large measure determines the form of the synaptic potential. Temporal summation of synaptic potential occurs during repetitive presynaptic stimulation, and after such stimulation the rate of decay of synaptic potential is greatly slowed.     

Molecular Mechanism of Active Zone Organization at Vertebrate Neuromuscular Junctions

Organization of presynaptic active zones is essential for development, plasticity, and pathology of the nervous system. Recent studies indicate a trans-synaptic molecular mechanism that organizes the active zones by connecting the pre- and the postsynaptic specialization. The presynaptic component of this trans-synaptic mechanism is comprised of cytosolic active zone proteins bound to the cytosolic domains of voltage-dependent calcium channels (P/Q-, N-, and L-type) on the presynaptic membrane. The postsynaptic component of this mechanism is the synapse organizer (laminin β2) that is expressed by the postsynaptic cell and accumulates specifically on top of the postsynaptic specialization. The pre- and the postsynaptic components interact directly between the extracellular domains of calcium channels and laminin β2 to anchor the presynaptic protein complex in front of the postsynaptic specialization. Hence, the presynaptic calcium channel functions as a scaffolding protein for active zone organization and as an ion-conducting channel for synaptic transmission. In contrast to the requirement of calcium influx for synaptic transmission, the formation of the active zone does not require the calcium influx through the calcium channels. Importantly, the active zones of adult synapses are not stable structures and require maintenance for their integrity. Furthermore, aging or diseases of the central and peripheral nervous system impair the active zones. This review will focus on the molecular mechanisms that organize the presynaptic active zones and summarize recent findings at the neuromuscular junctions and other synapses.