Stefan Hallermann

Maturation of active zone assembly by Drosophila Bruchpilot

Synaptic vesicles fuse at active zone (AZ) membranes where Ca2+ channels are clustered and that are typically decorated by electron-dense projections. Recently, mutants of the Drosophila melanogaster ERC/CAST family protein Bruchpilot (BRP) were shown to lack dense projections (T-bars) and to suffer from Ca2+ channel–clustering defects. In this study, we used high resolution light microscopy, electron microscopy, and intravital imaging to analyze the function of BRP in AZ assembly. Consistent with truncated BRP variants forming shortened T-bars, we identify BRP as a direct T-bar component at the AZ center with its N terminus closer to the AZ membrane than its C terminus. In contrast, Drosophila Liprin-α, another AZ-organizing protein, precedes BRP during the assembly of newly forming AZs by several hours and surrounds the AZ center in few discrete punctae. BRP seems responsible for effectively clustering Ca2+ channels beneath the T-bar density late in a protracted AZ formation process, potentially through a direct molecular interaction with intracellular Ca2+ channel domains.

Single Ih Channels in Pyramidal Neuron Dendrites: Properties, Distribution, and Impact on Action Potential Output

The hyperpolarization-activated cation current (Ih) plays an important role in regulating neuronal excitability, yet its native single-channel properties in the brain are essentially unknown. Here we use variance-mean analysis to study the properties of single Ih channels in the apical dendrites of cortical layer 5 pyramidal neurons in vitro. In these neurons, we find that Ih channels have an average unitary conductance of 680 ± 30 fS (n = 18). Spectral analysis of simulated and native Ih channels showed that there is little or no channel flicker below 5 kHz. In contrast to the uniformly distributed single-channel conductance, Ih channel number increases exponentially with distance, reaching densities as high as ∼550 channels/μm2 at distal dendritic sites. These high channel densities generate significant membrane voltage noise. By incorporating a stochastic model of Ih single-channel gating into a morphologically realistic model of a layer 5 neuron, we show that this channel noise is higher in distal dendritic compartments and increased threefold with a 10-fold increased single-channel conductance (6.8 pS) but constant Ih current density. In addition, we demonstrate that voltage fluctuations attributable to stochastic Ih channel gating impact on action potential output, with greater spike-timing precision in models with the experimentally determined single-channel conductance. These data suggest that, in the face of high current densities, the small single-channel conductance of Ih is critical for maintaining the fidelity of action potential output.

Stiff person syndrome-associated autoantibodies to amphiphysin mediate reduced GABAergic inhibition

Synaptic inhibition is a central factor in the fine tuning of neuronal activity in the central nervous system. Symptoms consistent with reduced inhibition such as stiffness, spasms and anxiety occur in paraneoplastic stiff person syndrome with autoantibodies against the intracellular synaptic protein amphiphysin. Here we show that intrathecal application of purified anti-amphiphysin immunoglobulin G antibodies induces stiff person syndrome-like symptoms in rats, including stiffness and muscle spasms. Using in vivo recordings of Hoffmann reflexes and dorsal root potentials, we identified reduced presynaptic GABAergic inhibition as an underlying mechanism. Anti-amphiphysin immunoglobulin G was internalized into neurons by an epitope-specific mechanism and colocalized in vivo with presynaptic vesicular proteins, as shown by stimulation emission depletion microscopy. Neurons from amphiphysin deficient mice that did not internalize the immunoglobulin provided additional evidence of the specificity in antibody uptake. GABAergic synapses appeared more vulnerable than glutamatergic synapses to defective endocytosis induced by anti-amphiphysin immunoglobulin G, as shown by increased clustering of the endocytic protein AP180 and by defective loading of FM 1-43, a styryl dye used to label cell membranes. Incubation of cultured neurons with anti-amphiphysin immunoglobulin G reduced basal and stimulated release of γ-aminobutyric acid substantially more than that of glutamate. By whole-cell patch-clamp analysis of GABAergic inhibitory transmission in hippocampus granule cells we showed a faster, activity-dependent decrease of the amplitude of evoked inhibitory postsynaptic currents in brain slices treated with antibodies against amphiphysin. We suggest that these findings may explain the pathophysiology of the core signs of stiff person syndrome at the molecular level and show that autoantibodies can alter the function of inhibitory synapses in vivo upon binding to an intraneuronal key protein by disturbing vesicular endocytosis.

Bassoon Speeds Vesicle Reloading at a Central Excitatory Synapse

Summary Sustained rate-coded signals encode many types of sensory modalities. Some sensory synapses possess specialized ribbon structures, which tether vesicles, to enable high-frequency signaling. However, central synapses lack these structures, yet some can maintain signaling over a wide bandwidth. To analyze the underlying molecular mechanisms, we investigated the function of the active zone core component Bassoon in cerebellar mossy fiber to granule cell synapses. We show that short-term synaptic depression is enhanced in Bassoon knockout mice during sustained high-frequency trains but basal synaptic transmission is unaffected. Fluctuation and quantal analysis as well as quantification with constrained short-term plasticity models revealed that the vesicle reloading rate was halved in the absence of Bassoon. Thus, our data show that the cytomatrix protein Bassoon speeds the reloading of vesicles to release sites at a central excitatory synapse.

Activity-dependent site-specific changes of glutamate receptor composition in vivo

The subunit composition of postsynaptic non–NMDA-type glutamate receptors (GluRs) determines the function and trafficking of the receptor. Changes in GluR composition have been implicated in the homeostasis of neuronal excitability and synaptic plasticity underlying learning. Here, we imaged GluRs in vivo during the formation of new postsynaptic densities (PSDs) at Drosophila neuromuscular junctions coexpressing GluRIIA and GluRIIB subunits. GluR composition was independently regulated at directly neighboring PSDs on a submicron scale. Immature PSDs typically had large amounts of GluRIIA and small amounts of GluRIIB. During subsequent PSD maturation, however, the GluRIIA/GluRIIB composition changed and became more balanced. Reducing presynaptic glutamate release increased GluRIIA, but decreased GluRIIB incorporation. Moreover, the maturation of GluR composition correlated in a site-specific manner with the level of Bruchpilot, an active zone protein that is essential for mature glutamate release. Thus, we show that an activity-dependent, site-specific control of GluR composition can contribute to match pre- and postsynaptic assembly.

A large pool of releasable vesicles in a cortical glutamatergic synapse

To probe exocytosis at a cortical glutamatergic synapse, we made capacitance measurements in whole-cell recorded hippocampal mossy fiber terminals. Evaluation of different methods by using a morphology-based equivalent electrical model revealed that quantitative capacitance measurements are possible in this presynaptic structure. Voltage pulses leading to presynaptic Ca2+ inflow evoked large capacitance signals that showed saturation with increasing pulse duration. The mean peak capacitance increase was 100 fF, corresponding to a pool of ≈1,400 releasable vesicles. Thus hippocampal mossy fiber synapses have a vesicular “maxipool.” Large pool size and rapid vesicle recycling may underlie the uniquely large extent of activity-dependent plasticity in this synapse.

Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states

The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. Here we introduce new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ cytomatrix (CAZ) is composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. We test for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure.

Rapid Active Zone Remodeling during Synaptic Plasticity

How can synapses change the amount of neurotransmitter released during synaptic plasticity? Although release in general is intensely investigated, its determinants during plasticity are still poorly understood. As a model for plastic strengthening of synaptic release, we here use the well-established presynaptic homeostatic compensation during interference with postsynaptic glutamate receptors at the Drosophila neuromuscular junction. Combining short-term plasticity analysis, cumulative EPSC analysis, fluctuation analysis, and quantal short-term plasticity modeling, we found an increase in the number of release-ready vesicles during presynaptic strengthening. High-resolution light microscopy revealed an increase in the amount of the active zone protein Bruchpilot and an enlargement of the presynaptic cytomatrix structure. Furthermore, these functional and structural alterations of the active zone were not only observed after lifelong but already after minutes of presynaptic strengthening. Our results demonstrate that presynaptic plasticity can induce active zone remodeling, which regulates the number of release-ready vesicles within minutes.

Naked dense bodies provoke depression.

At presynaptic active zones (AZs), the frequently observed tethering of synaptic vesicles to an electron-dense cytomatrix represents a process of largely unknown functional significance. Here, we identified a hypomorphic allele, brpnude, lacking merely the last 1% of the C-terminal amino acids (17 of 1740) of the active zone protein Bruchpilot. In brpnude, electron-dense bodies were properly shaped, though entirely bare of synaptic vesicles. While basal glutamate release was unchanged, paired-pulse and sustained stimulation provoked depression. Furthermore, rapid recovery following sustained release was slowed. Our results causally link, with intramolecular precision, the tethering of vesicles at the AZ cytomatrix to synaptic depression.

State and location dependence of action potential metabolic cost in cortical pyramidal neurons

Action potential generation and conduction requires large quantities of energy to restore Na+ and K+ ion gradients. We investigated the subcellular location and voltage dependence of this metabolic cost in rat neocortical pyramidal neurons. Using Na+/K+ charge overlap as a measure of action potential energy efficiency, we found that action potential initiation in the axon initial segment (AIS) and forward propagation into the axon were energetically inefficient, depending on the resting membrane potential. In contrast, action potential backpropagation into dendrites was efficient. Computer simulations predicted that, although the AIS and nodes of Ranvier had the highest metabolic cost per membrane area, action potential backpropagation into the dendrites and forward propagation into axon collaterals dominated energy consumption in cortical pyramidal neurons. Finally, we found that the high metabolic cost of action potential initiation and propagation down the axon is a trade-off between energy minimization and maximization of the conduction reliability of high-frequency action potentials.