Dynamics underlying synaptic gain between pairs of cortical pyramidal neurons

Changes in connectivity between pairs of neurons can serve as a substrate for information storage and for experience-dependent changes in neuronal circuitry. Early in development, synaptic contacts form and break, but how these dynamics influence the connectivity between pairs of neurons is not known. Here we used time-lapse imaging to examine the synaptic interactions between pairs of cultured cortical pyramidal neurons, and found that the axon-dendrite contacts between each neuronal pair were composed of both a relatively stable and a more labile population. Under basal conditions, loss and gain of contacts within this labile population was well balanced and there was little net change in connectivity. Selectively increasing the levels of activated CaMKII in the postsynaptic neuron increased connectivity between pairs of neurons by increasing the rate of gain of new contacts without affecting the probability of contact loss, or the proportion of stable and labile contacts, and this increase required Calcium/calmodulin binding to CaMKII. Our data suggest that activating CaMKII can increase synaptic connectivity through a CaM-dependent increase in contact formation, followed by stabilization of a constant fraction of new contacts.     

Immunoisolation of two synaptic vesicle pools from synaptosomes: a proteomics analysis

The nerve terminal proteome governs neurotransmitter release as well as the structural and functional dynamics of the presynaptic compartment. In order to further define specific presynaptic subproteomes we used subcellular fractionation and a monoclonal antibody against the synaptic vesicle protein SV2 for immunoaffinity purification of two major synaptosome-derived synaptic vesicle-containing fractions: one sedimenting at lower and one sedimenting at higher sucrose density. The less dense fraction contains free synaptic vesicles, the denser fraction synaptic vesicles as well as components of the presynaptic membrane compartment. These immunoisolated fractions were analyzed using the cationic benzyldimethyl-n-hexadecylammonium chloride (BAC) polyacrylamide gel system in the first and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Protein spots were subjected to analysis by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS). We identified 72 proteins in the free vesicle fraction and 81 proteins in the plasma membrane-containing denser fraction. Synaptic vesicles contain a considerably larger number of protein constituents than previously anticipated. The plasma membrane-containing fraction contains synaptic vesicle proteins, components of the presynaptic fusion and retrieval machinery and numerous other proteins potentially involved in regulating the functional and structural dynamics of the nerve terminal.     

Key role of calcium signaling in synaptic transmission

The review analyzes current concepts on the role of calcium signals in the process of synaptic neuron-to-neuron and neuron-effector transmission and on the respective intracellular mechanisms (participation of different types of ion channels, mitochondria, and endoplasmic reticulum). The involvement of calcium both in the transmission per se, in particular in the process of exocytotic neurotransmitter release, and in long-lasting modulations of the efficacy of synaptic transmission (potentiation or depression) is considered. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 290–293, July–October, 2007.     

长时程突触可塑性中的突触标识

神经元长时程突触可塑性是学习和记忆的基础,神经元长时程突触可塑性的维持依赖于基因的转录和蛋白质合成.然而,这些转录产物和新合成的蛋白质是如何从胞体运输到突触点,还不甚清楚.近年来的研究显示,当长时程突触可塑性发生时,被激活的突触能通过建立突触标记(synaptic tag)来识别、捕捉和利用其所需要的基因产物,以维持突触可塑性的长时程变化.这一过程或现象被称为突触标识(synaptic tagging).本文就近年来突触标识的研究进展作一概述.     

Membrane topology of the Drosophila vesicular glutamate transporter

The vesicular glutamate transporters (VGLUTs) are responsible for packaging glutamate into synaptic vesicles, and are part of a family of structurally related proteins that mediate organic anion transport. Standard computer-based predictions of transmembrane domains have led to divergent topological models, indicating the need for experimentally derived predictions. Here we present data on the topology of the VGLUT ortholog from Drosophila melanogaster (DVGLUT). Using immunofluorescence assays of DVGLUT transiently localized to the plasma membrane of heterologously transfected cells, we have determined the accessibility of epitope tags inserted into the lumenal/extracellular face of the protein. Using immunoisolation, we have identified complementary tagged sites that face the cytoplasm. Our data show that DVGLUT contains 10 hydrophobic regions that completely span the membrane (TMs 1-10) and that the amino and carboxyl termini are cytosolic. Importantly, between TMs 4 and 5 is an unforeseen cytosolic loop of some 50 residues. Other domains exposed to the cytosol include loops between TMs 6-7 and 8-9, and regions C-terminal to TM2 and N-terminal to TM3. Between TM2 and 3 is a potentially hydrophobic, but topologically ambiguous region. Lumenal domains include sequences between TMs 1-2, 3-4, 5-6, 7-8 and 9-10. These data provide a basis for determining structure-function relationships for DVGLUT and other related proteins.     

Developmental and activity-dependent regulation of ARMS/Kidins220 in cultured rat hippocampal neurons

Neurotrophin activation of Trk receptors elicits diverse effects on neuronal survival, differentiation, and synaptic plasticity. One of the central questions is how specificity is encoded in neurotrophin receptor signaling and actions. A unique downstream protein is the Ankyrin-Repeat Rich Membrane Spanning (ARMS)/Kinase D-interacting substrate-220 kDa (Kidins220), a very abundant scaffold protein in the hippocampus. To determine the roles of ARMS/Kidins220 in hippocampal neurons, we have analyzed the effects of synaptic activity upon the regulation and distribution of ARMS/Kidins220. At early times in vitro (<7 DIV), synaptic activity was low and ARMS/Kidins220 levels were high. As synaptic activity and markers for synapse maturation, such as PSD-95, increased, ARMS/Kidins220 significantly decreased to a plateau by later times in vitro (>12 DIV). Immunocytochemistry showed ARMS/Kidins220 to be concentrated at the tips of growing processes in immature cultures, and more diffusely distributed in older cultures. To examine the apparent inverse relationship between activity and ARMS/Kidins220 levels, neuronal firing was manipulated pharmacologically. Chronic exposure to TTX increased ARMS/Kidins220 levels, whereas bicuculline caused the opposite effect. Moreover, using shRNA to decrease ARMS/Kidins220 levels produced a corresponding increase in synaptic activity. We find that ARMS/Kidins220 may function in neuronal development as an indicator and potentially as a homeostatic regulator of overall synaptic strength in hippocampal neurons.     

Identification and characterization of SV31, a novel synaptic vesicle membrane protein and potential transporter

Synaptic vesicle proteins govern all relevant functions of the synaptic vesicle life cycle, including vesicle biogenesis, vesicle transport, uptake and storage of neurotransmitters, and regulated endocytosis and exocytosis. In spite of impressive progress made in the past years, not all known vesicular functions can be assigned to defined protein components, suggesting that the repertoire of synaptic vesicle proteins is still incomplete. We have identified and characterized a novel synaptic vesicle membrane protein of 31 kDa with six putative transmembrane helices that, according to its membrane topology and phylogenetic relation, may function as a vesicular transporter. The vesicular allocation is demonstrated by subcellular fractionation, heterologous expression, immunocytochemical analysis of brain sections and immunoelectron microscopy. The protein is expressed in select brain regions and contained in subpopulations of nerve terminals that immunostain for the vesicular glutamate transporter 1 and the vesicular GABA transporter VGaT (vesicular amino acid transporter) and may attribute specific and as yet undiscovered functions to subsets of glutamatergic and GABAergic synapses.     

Local sleep homeostasis in the avian brain: convergence of sleep function in mammals and birds?

The function of the brain activity that defines slow wave sleep (SWS) and rapid eye movement (REM) sleep in mammals is unknown. During SWS, the level of electroencephalogram slow wave activity (SWA or 0.5-4.5 Hz power density) increases and decreases as a function of prior time spent awake and asleep, respectively. Such dynamics occur in response to waking brain use, as SWA increases locally in brain regions used more extensively during prior wakefulness. Thus, SWA is thought to reflect homeostatically regulated processes potentially tied to maintaining optimal brain functioning. Interestingly, birds also engage in SWS and REM sleep, a similarity that arose via convergent evolution, as sleeping reptiles and amphibians do not show similar brain activity. Although birds deprived of sleep show global increases in SWA during subsequent sleep, it is unclear whether avian sleep is likewise regulated locally. Here, we provide, to our knowledge, the first electrophysiological evidence for local sleep homeostasis in the avian brain. After staying awake watching David Attenborough's The Life of Birds with only one eye, SWA and the slope of slow waves (a purported marker of synaptic strength) increased only in the hyperpallium--a primary visual processing region--neurologically connected to the stimulated eye. Asymmetries were specific to the hyperpallium, as the non-visual mesopallium showed a symmetric increase in SWA and wave slope. Thus, hypotheses for the function of mammalian SWS that rely on local sleep homeostasis may apply also to birds.     

Shank mutant mice as an animal model of autism

In this review, we focus on the role of the Shank family of proteins in autism. In recent years, autism research has been flourishing. With genetic, molecular, imaging and electrophysiological studies being supported by behavioural studies using animal models, there is real hope that we may soon understand the fundamental pathology of autism. There is also genuine potential to develop a molecular-level pharmacological treatment that may be able to deal with the most severe symptoms of autism, and clinical trials are already underway. The Shank family of proteins has been strongly implicated as a contributing factor in autism in certain individuals and sits at the core of the alleged autistic pathway. Here, we analyse studies that relate Shank to autism and discuss what light this sheds on the possible causes of autism.     

Spatial Localization of Synapses Required for Supralinear Summation of Action Potentials and EPSPs

Although the supralinear summation of synchronizing excitatory postsynaptic potentials (EPSPs) and backpropagating action potentials (APs) is important for spike-timing-dependent synaptic plasticity (STDP), the spatial conditions of the amplification in the divergent dendritic structure have yet to be analyzed. In the present study, we simulated the coincidence of APs with EPSPs at randomly determined synaptic sites of a morphologically reconstructed hippocampal CA1 pyramidal model neuron and clarified the spatial condition of the amplifying synapses. In the case of uniform conductance inputs, the amplifying synapses were localized in the middle apical dendrites and distal basal dendrites with small diameters, and the ratio of synapses was unexpectedly small: 8-16% in both apical and basal dendrites. This was because the appearance of strong amplification requires the coincidence of both APs of 3-30 mV and EPSPs of over 6 mV, both of which depend on the dendritic location of synaptic sites. We found that the localization of amplifying synapses depends on A-type K+ channel distribution because backpropagating APs depend on the A-type K+ channel distribution, and that the localizations of amplifying synapses were similar within a range of physiological synaptic conductances. We also quantified the spread of membrane amplification in dendrites, indicating that the neighboring synapses can also show the amplification. These findings allowed us to computationally illustrate the spatial localization of synapses for supralinear summation of APs and EPSPs within thin dendritic branches where patch clamp experiments cannot be easily conducted.     

SV31 is a Zn2+-binding synaptic vesicle protein

We recently identified in a proteomic screen a novel synaptic vesicle membrane protein of 31 kDa (SV31) of unknown function. According to its membrane topology and its phylogenetic relation SV31 may function as a vesicular transporter. Based on its amino acid sequence similarity to a prokaryotic heavy metal ion transporter we analyzed its metal ion-binding properties and show that recombinant SV31 binds the divalent cations Zn(2+) and Ni(2+) and to a minor extent Cu(2+), but not Fe(2+), Co(2+), Mn(2+), or Ca(2+). Zn(2+)-binding of SV31 in viable cells was verified following heterologous transfection of pheochromocytoma cells 12 (PC12) with recombinant red fluorescent SV31 (SV31-RFP) and the fluorescent zinc indicator FluoZin-3. Sucrose density gradient fractionation of SV31-RFP-transfected PC12 cells revealed a partial overlap of SV31-RFP with synaptic-like vesicle markers and the early endosome marker rab5. Immunocytochemical analysis demonstrated a punctuate distribution in the cell soma and in neuritic processes and in addition in a compartment in vicinity to the plasma membrane that was immunopositive also for synaptosomal-associated protein 25 (SNAP-25) and syntaxin1A. Our data suggest that SV31 represents a novel Zn(2+) -binding protein that in PC12 cells is targeted to endosomes and subpopulations of synaptic-like microvesicles.     

Acute Complexin Knockout Abates Spontaneous and Evoked Transmitter Release

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Dynamic modification of dendritic cable properties and synaptic transmission by voltage-gated potassium channels

Computer simulations of a dendrite possessing voltage-sensitive potassium conductances were used to determine the effects of these conductances on synaptic transmission and on the propagation of synaptic signals within the dendritic tree. Potassium conductances had two principal effects on voltage transients generated by current injections or synaptic conductances. Locally (near the source of the transient), voltage-gated potassium channels produced a potassium shunt current that reduced the amplitude of voltage transients generated by depolarizing currents. This shunt current increased as the amplitude of the depolarizing transient increased and so acted to prevent large synaptic transients from reaching levels that would saturate due to a reduction in driving force. In the presence of rapidly activating potassium currents, excitatory synapses produced larger synaptic currents that were more linearly related to synaptic conductance, but these produced smaller voltage transients. The maximum amplitudes of the voltage transients were limited by the voltage sensitivity of the K⁺ conductance and the rate at which it could activate. Sufficiently rapid synaptic currents could outrun the K⁺ conductance and thus achieve high local peak amplitudes. These effects of K⁺ conductances were unrelated to whether they were located on dendrites or not, being related only to their proximity to the source of synaptic current. The second class of effects of K⁺ conductances depended on their alteration of the electrotonic structure of the postsynaptic cell and so were observed only when they were located on postsynaptic dendrites. Voltage-gated K⁺ conductances produced voltage-dependent electrotonic expansion of depolarized dendrites, which had the effect of isolating synaptic inputs on depolarized dendrites from events on the rest of the neuron. Thus, synapses on the same dendrite interacted destructively to a degree much greater than that expected from the classical driving force nonlinearity. Synapses located proximally to a depolarized dendritic region were less effected than those located distally, and the range of the nonlinear interaction between synapses was dependent on the kinetics of activation and deactivation of the conductance. When present in conjunction with rapidly activating dendritic sodium conductance, the potassium conductance sharpened the requirement for spatial and temporal coincidence to produce synaptic boosting by inward currents, and suppressed out-of-synchrony synaptic inputs.     

Synaptic repression at crayfish neuromuscular junctions. I. Generation after partial target area removal

Synaptic repression, the inability of synaptic junctions to generate normal-sized postsynaptic potentials under normal physiological conditions, is reported here for crayfish neuromuscular synapses. The synapses in the superficial flexor muscle system of the crayfish change their efficiency in generating a postsynaptic response as a result of a specific alteration in their immediate environment. When the superficial flexor nerve is cut halfway into the target muscle field and the lateral muscle fibers are removed, the intact medial synapses do not generate normal-sized junction potentials (JP) at the 17° –19°C temperature of the Ringers solution. JPs cannot be recorded in 83% of the muscle fibers at 2 weeks after the operation and of the few JPs that can be detected, 80% are smaller than 1 mV in size. By 8 weeks after the operation, JPs were detected in 55% of the muscle fibers, and now only 46% of these are smaller than 1 mV. When the lateral muscle fibers are left in place during the original operation, providing a target area for the cut nerve to grow into, JPs were then detected in 60%–80% of all medial fibers at all time periods after the operation; their size profile, with 10%–25% of the muscle fibers having JP's less than 1 mV, was similar to control values. These results suggest that the efficiency of these synaptic contacts become affected as a result of partial axotomy and removal of the target area of the cut branches of the axons. © 1993 John Wiley & Sons, Inc.     

The Drosophila epsin 1 is required for ubiquitin-dependent synaptic growth and function but not for synaptic vesicle recycling

The ubiquitin-proteasome system plays an important role in synaptic development and function. However, many components of this system, and how they act to affect synapses, are still not well understood. In this study, we use the Drosophila neuromuscular junction to study the in vivo function of Liquid facets (Lqf), a homolog of mammalian epsin 1. Our data show that Lqf plays a novel role in synapse development and function. Contrary to prior models, Lqf is not required for clathrin-mediated endocytosis of synaptic vesicles. Lqf is required to maintain bouton size and shape and to sustain synapse growth by acting as a specific substrate of the deubiquitinating enzyme Fat facets. However, Lqf is not a substrate of the Highwire (Hiw) E3 ubiquitin ligase; neither is it required for synapse overgrowth in hiw mutants. Interestingly, Lqf converges on the Hiw pathway by negatively regulating transmitter release in the hiw mutant. These observations demonstrate that Lqf plays distinct roles in two ubiquitin pathways to regulate structural and functional plasticity of the synapse.