Christoph Körber

Postsynaptic Neuroligin1 regulates presynaptic maturation

Presynaptic nerve terminals pass through distinct stages of maturation after their initial assembly. Here we show that the postsynaptic cell adhesion molecule Neuroligin1 regulates key steps of presynaptic maturation. Presynaptic terminals from Neuroligin1-knockout mice remain structurally and functionally immature with respect to active zone stability and synaptic vesicle pool size, as analyzed in cultured hippocampal neurons. Conversely, overexpression of Neuroligin1 in immature neurons, that is within the first 5 days after plating, induced the formation of presynaptic boutons that had hallmarks of mature boutons. In particular, Neuroligin1 enhanced the size of the pool of recycling synaptic vesicles, the rate of synaptic vesicle exocytosis, the fraction of boutons responding to depolarization, as well as the responsiveness of the presynaptic release machinery to phorbol ester stimulation. Moreover, Neuroligin1 induced the formation of active zones that remained stable in the absence of F-actin, another hallmark of advanced maturation. Acquisition of F-actin independence of the active zone marker Bassoon during culture development or induced via overexpression of Neuroligin1 was activity-dependent. The extracellular domain of Neuroligin1 was sufficient to induce assembly of functional presynaptic terminals, while the intracellular domain was required for terminal maturation. These data show that induction of presynaptic terminal assembly and maturation involve mechanistically distinct actions of Neuroligins, and that Neuroligin1 is essential for presynaptic terminal maturation.

A small pool of vesicles maintains synaptic activity in vivo

Chemical synapses contain substantial numbers of neurotransmitter-filled synaptic vesicles, ranging from approximately 100 to many thousands. The vesicles fuse with the plasma membrane to release neurotransmitter and are subsequently reformed and recycled. Stimulation of synapses in vitro generally causes the majority of the synaptic vesicles to release neurotransmitter, leading to the assumption that synapses contain numerous vesicles to sustain transmission during high activity. We tested this assumption by an approach we termed cellular ethology, monitoring vesicle function in behaving animals (10 animal models, nematodes to mammals). Using FM dye photooxidation, pHluorin imaging, and HRP uptake we found that only approximately 1–5% of the vesicles recycled over several hours, in both CNS synapses and neuromuscular junctions. These vesicles recycle repeatedly, intermixing slowly (over hours) with the reserve vesicles. The latter can eventually release when recycling is inhibited in vivo but do not seem to participate under normal activity. Vesicle recycling increased only to ≈5% in animals subjected to an extreme stress situation (frog predation on locusts). Synapsin, a molecule binding both vesicles and the cytoskeleton, may be a marker for the reserve vesicles: the proportion of vesicles recycling in vivo increased to 30% in synapsin-null Drosophila. We conclude that synapses do not require numerous reserve vesicles to sustain neurotransmitter release and thus may use them for other purposes, examined in the accompanying paper.

Electrophysiological Properties of AMPA Receptors Are Differentially Modulated Depending on the Associated Member of the TARP Family

The family of AMPA receptors is encoded by four genes that are differentially spliced to result in the flip or flop versions of the four subunits GluR1 to GluR4. GluR2 is further modified at the so-called Q/R site by posttranscriptional RNA editing. Delivery of AMPA receptors to the plasma membrane and synaptic trafficking are controlled by transmembrane AMPA receptor regulatory proteins (TARPs). Additionally, TARPs influence essential electrophysiological properties of AMPA receptor channels such as desensitization and agonist efficacies. Here, we compare the influence of all known TARPs (γ2, γ3, γ4, and γ8) on agonist-induced currents of the four AMPA receptor subunits, including flip and flop splice variants and editing variants. We show that, although agonist-induced currents of all homomeric AMPA receptor subunits as well as all heteromeric combinations tested are significantly potentiated when coexpressed with members of the TARP family in Xenopus laevis oocytes, the extent of TARP-mediated increase in agonist-induced responses is highly dependent on both the AMPA receptor subunit and the coexpressed TARP. Moreover, we demonstrate that the splice variant of the AMPA receptor plays a key role in determining the modulation of electrophysiological properties by associated TARPs. We furthermore present evidence that individual TARP–AMPA receptor interactions control the degree of desensitization of AMPA receptors. Consequently, because of their subunit-specific impact on the electrophysiological properties, TARPs play a major role as modulatory subunits of AMPA receptors and thus contribute to the functional diversity of AMPA receptors encountered in the CNS.

Serial section scanning electron microscopy (S3EM) on silicon wafers for ultra-structural volume imaging of cells and tissues.

High resolution, three-dimensional (3D) representations of cellular ultrastructure are essential for structure function studies in all areas of cell biology. While limited subcellular volumes have been routinely examined using serial section transmission electron microscopy (ssTEM), complete ultrastructural reconstructions of large volumes, entire cells or even tissue are difficult to achieve using ssTEM. Here, we introduce a novel approach combining serial sectioning of tissue with scanning electron microscopy (SEM) using a conductive silicon wafer as a support. Ribbons containing hundreds of 35 nm thick sections can be generated and imaged on the wafer at a lateral pixel resolution of 3.7 nm by recording the backscattered electrons with the in-lens detector of the SEM. The resulting electron micrographs are qualitatively comparable to those obtained by conventional TEM. S3EM images of the same region of interest in consecutive sections can be used for 3D reconstructions of large structures. We demonstrate the potential of this approach by reconstructing a 31.7 µm3 volume of a calyx of Held presynaptic terminal. The approach introduced here, Serial Section SEM (S3EM), for the first time provides the possibility to obtain 3D ultrastructure of large volumes with high resolution and to selectively and repetitively home in on structures of interest. S3EM accelerates process duration, is amenable to full automation and can be implemented with standard instrumentation.

The Transmembrane AMPA Receptor Regulatory Protein γ4 Is a More Effective Modulator of AMPA Receptor Function than Stargazin (γ2)

AMPA receptors mediate the majority of the fast excitatory synaptic transmission in the brain. A family of recently described auxiliary proteins, the transmembrane AMPA receptor regulatory proteins (TARPs) γ2, γ3, γ4, and γ8, have been shown to modulate the trafficking of receptors to the plasma membrane as well as electrophysiological key properties. Most studies published to date focus exclusively on γ2 (stargazin), neglecting the other three members of the TARP family. Here, we analyzed the modulation of electrophysiological properties of AMPA receptors by γ4 and compare it with γ2, using heterologous coexpression in human embryonic kidney 293 cells. We show for the first time that γ4, a previously poorly examined TARP, modulates the desensitization properties of AMPA receptors significantly stronger than γ2 does. In contrast, other properties such as kainate efficacy and current–voltage relationships are modulated in a similar way by both of these TARPs. From these TARP-specific effects, we propose an interaction mechanism between AMPA receptors and TARPs and address the physiological relevance of γ4 and its regulatory effects, particularly on AMPA receptor desensitization properties, to developmental and regulatory processes in the brain.

Stargazin Interaction with α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionate (AMPA) Receptors Is Critically Dependent on the Amino Acid at the Narrow Constriction of the Ion Channel

The subunit GluR2 of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) subfamily of ionotropic glutamate receptors (GluRs) features a single amino acid at the narrow constriction of the pore loop that is altered from glutamine to arginine by RNA editing. This so-called Q/R site has been shown to play an important role in the determination of the electrophysiological properties of AMPA receptor complexes as well as of trafficking to the plasma membrane. The protein stargazin has also been shown to modulate electrophysiological properties and trafficking to the plasma membrane of AMPA receptors. In this study we examined via a series of mutants of the Q/R site of the AMPA receptor GluR1 whether the amino acid at this position has any influence on the modulatory effects mediated by stargazin. To this end, we analyzed current responses of Q/R site mutants upon application of glutamate and kainate and determined the amount of mutant receptor protein in the plasma membrane in Xenopus oocytes. Desensitization kinetics of several mutants were analyzed in HEK293 cells. We found that the stargazin-mediated decrease in receptor desensitization, the slowing of desensitization kinetics, and the kainate efficacy were all dependent on the amino acid at the Q/R site, whereas the stargazin-mediated increase in trafficking toward the plasma membrane remained independent of this amino acid. We propose that the Q/R site modulates the interaction of stargazin with the transmembrane domains of AMPA receptors via an allosteric mechanism and that this modulation leads to the observed differences in the electrophysiological properties of the receptor.

Molecular machines regulating the release probability of synaptic vesicles at the active zone.

The fusion of synaptic vesicles (SVs) with the plasma membrane of the active zone (AZ) upon arrival of an action potential (AP) at the presynaptic compartment is a tightly regulated probabilistic process crucial for information transfer. The probability of a SV to release its transmitter content in response to an AP, termed release probability (Pr), is highly diverse both at the level of entire synapses and individual SVs at a given synapse. Differences in Pr exist between different types of synapses, between synapses of the same type, synapses originating from the same axon and even between different SV subpopulations within the same presynaptic terminal. The Pr of SVs at the AZ is set by a complex interplay of different presynaptic properties including the availability of release-ready SVs, the location of the SVs relative to the voltage-gated calcium channels (VGCCs) at the AZ, the magnitude of calcium influx upon arrival of the AP, the buffering of calcium ions as well as the identity and sensitivity of the calcium sensor. These properties are not only interconnected, but can also be regulated dynamically to match the requirements of activity patterns mediated by the synapse. Here, we review recent advances in identifying molecules and molecular machines taking part in the determination of vesicular Pr at the AZ.

A Domain Linking the AMPA Receptor Agonist Binding Site to the Ion Pore Controls Gating and Causes lurcher Properties when Mutated

Ionotropic, AMPA-type glutamate receptors (GluRs) critically shape excitatory synaptic signals in the CNS. Ligand binding induces conformational changes in the glutamate-binding domain of the receptors that are converted into opening of the channel pore via three short linker sequences, a process referred to as gating. Although crystallization of the glutamate-binding domain and structural models of the ion pore advanced our understanding of ligand-binding dynamics and pore movements, the allosteric coupling of both events by the short linkers has not been described in detail. To study the role of the linkers in gating GluR1, we transplanted them between different GluRs and examined the electrophysiological properties of the resulting chimeric receptors in Xenopus laevis oocytes and HEK293 cells. We found that all three linkers decisively affect receptor functionality, agonist potency, and desensitization. One linker chimera was nondesensitizing and exhibited strongly increased agonist potencies, while fluxing ions even in the absence of agonist, similar to properties reported for the GluR1 lurcher mutation. Combining this new lurcher-like linker chimera with the original lurcher mutation allowed us to reassess the effect of lurcher on GluR1 gating properties. The observed differential but interdependent influence of linker and lurcher mutations on receptor properties suggests that the linkers are part of a fine-tuned structural element that normally stabilizes the closed ion pore. We propose that lurcher-like mutations act by disrupting this element such that ligand-induced conformational changes are not necessarily required to gate the channel.

Effects of distinct collybistin isoforms on the formation of GABAergic synapses in hippocampal neurons.

Collybistin (Cb) is a brain specific guanine nucleotide exchange factor that interacts with the inhibitory postsynaptic scaffold protein gephyrin. Cb is essential for the postsynaptic clustering of gephyrin and major GABA(A) receptor subtypes during the formation and maintenance of GABAergic synapses in the hippocampus and other areas of the forebrain. In the rat, four distinct splice variants (Cb1, Cb2(SH3-), Cb2(SH3+) and Cb3), have been described, which differ in their C-termini (Cb1-3) and in respect of the SH3-domain that is absent in Cb2(SH3-). In the human brain, only a single isoform (hPEM2) corresponding to Cb3, was found to be expressed. This has been implicated in neurological defects such as hyperekplexia, epilepsy, anxiety, aggression and mental retardation. In this study, we address the functional significance of the differentially spliced Cb isoforms by generating a shRNA-mediated knock-down of endogenous Cb in hippocampal cultured neurons that is subsequently rescued by the expression of distinct Cb isoforms. We found that the Cb knock-down induced impairment in GABAergic neurotransmission could be rescued by the expression of any of the Cb isoforms, independent of their C-termini or the presence of the SH3-domain in the N-terminal region. Thus, the different Cb isoforms all confer basic functionality.

Endocytic Structures and Synaptic Vesicle Recycling at a Central Synapse in Awake Rats

The synaptic vesicle (SV) cycle has been studied extensively in cultured cells and slice preparations, but not much is known about the roles and relative contributions of endocytic pathways and mechanisms of SV recycling in vivo, under physiological patterns of activity. We employed horseradish peroxidase (HRP) as an in vivo marker of endocytosis at the calyx of Held synapse in the awake rat. Ex vivo serial section scanning electron microscopy and 3D reconstructions revealed two categories of labelled structures: HRP‐filled SVs and large cisternal endosomes. Inhibition of adaptor protein complexes 1 and 3 (AP‐1, AP‐3) by in vivo application of Brefeldin A (BFA) disrupted endosomal SV budding while SV recycling via clathrin‐mediated endocytosis (CME) remained unaffected. In conclusion, our study establishes cisternal endosomes as an intermediate of the SV cycle and reveals CME and endosomal budding as the predominant mechanisms of SV recycling in a tonically active central synapse in vivo.