U. Valentin Nägerl

Bidirectional Activity-Dependent Morphological Plasticity in Hippocampal Neurons

Dendritic spines on pyramidal neurons receive the vast majority of excitatory input and are considered electrobiochemical processing units, integrating and compartmentalizing synaptic input. Following synaptic plasticity, spines can undergo morphological plasticity, which possibly forms the structural basis for long-term changes in neuronal circuitry. Here, we demonstrate that spines on CA1 pyramidal neurons from organotypic slice cultures show bidirectional activity-dependent morphological plasticity. Using two-photon time-lapse microscopy, we observed that low-frequency stimulation induced NMDA receptor-dependent spine retractions, whereas theta burst stimulation led to the formation of new spines. Moreover, without stimulation the number of spine retractions was on the same order of magnitude as the stimulus-induced spine gain or loss. Finally, we found that the ability of neurons to eliminate spines in an activity-dependent manner decreased with developmental age. Taken together, our data show that hippocampal neurons can undergo bidirectional morphological plasticity; spines are formed and eliminated in an activity-dependent way.

Live-cell imaging of dendritic spines by STED microscopy

Time lapse fluorescence imaging has become one of the most important approaches in neurobiological research. In particular, both confocal and two-photon microscopy have been used to study activity-dependent changes in synaptic morphology. However, the diffraction-limited resolution of light microscopy is often inadequate, forcing researchers to complement the live cell imaging strategy by EM. Here, we report on the first use of a far-field optical technique with subdiffraction resolution to noninvasively image activity-dependent morphological plasticity of dendritic spines. Specifically we show that time lapse stimulated emission depletion imaging of dendritic spines of YFP-positive hippocampal neurons in organotypic slices outperforms confocal microscopy in revealing important structural details. The technique substantially improves the quantification of morphological parameters, such as the neck width and the curvature of the heads of spines, which are thought to play critical roles for the function and plasticity of synaptic connections.

A Balance of Protein Synthesis and Proteasome-Dependent Degradation Determines the Maintenance of LTP

Long-lasting changes in synaptic strength are thought to play a pivotal role in activity-dependent plasticity and memory. There is ample evidence indicating that in hippocampal long-term potentiation (LTP) the synthesis of new proteins is crucial for enduring changes. However, whether protein degradation also plays a role in this process has only recently begun to receive attention. Here, we examine the effects of blocking protein degradation on LTP. We show that pharmacological inhibition of proteasome-dependent protein degradation, just like inhibition of protein synthesis, disrupts expression of late (L-)LTP. However, when protein degradation and protein synthesis are inhibited at the same time, LTP is restored to control levels, calling into question the commonly held hypothesis that synthesis of new proteins is indispensable for L-LTP. Instead, these findings point to a more facetted model, in which L-LTP is determined by the combined action of synthesis and degradation of plasticity proteins.

Spine neck plasticity regulates compartmentalization of synapses

Dendritic spines have been proposed to transform synaptic signals through chemical and electrical compartmentalization. However, the quantitative contribution of spine morphology to synapse compartmentalization and its dynamic regulation are still poorly understood. We used time-lapse super-resolution stimulated emission depletion (STED) imaging in combination with fluorescence recovery after photobleaching (FRAP) measurements, two-photon glutamate uncaging, electrophysiology and simulations to investigate the dynamic link between nanoscale anatomy and compartmentalization in live spines of CA1 neurons in mouse brain slices. We report a diversity of spine morphologies that argues against common categorization schemes and establish a close link between compartmentalization and spine morphology, wherein spine neck width is the most critical morphological parameter. We demonstrate that spine necks are plastic structures that become wider and shorter after long-term potentiation. These morphological changes are predicted to lead to a substantial drop in spine head excitatory postsynaptic potential (EPSP) while preserving overall biochemical compartmentalization.

STED nanoscopy of actin dynamics in synapses deep inside living brain slices

It is difficult to investigate the mechanisms that mediate long-term changes in synapse function because synapses are small and deeply embedded inside brain tissue. Although recent fluorescence nanoscopy techniques afford improved resolution, they have so far been restricted to dissociated cells or tissue surfaces. However, to study synapses under realistic conditions, one must image several cell layers deep inside more-intact, three-dimensional preparations that exhibit strong light scattering, such as brain slices or brains in vivo. Using aberration-reducing optics, we demonstrate that it is possible to achieve stimulated emission depletion superresolution imaging deep inside scattering biological tissue. To illustrate the power of this novel (to our knowledge) approach, we resolved distinct distributions of actin inside dendrites and spines with a resolution of 60-80 nm in living organotypic brain slices at depths up to 120 μm. In addition, time-lapse stimulated emission depletion imaging revealed changes in actin-based structures inside spines and spine necks, and showed that these dynamics can be modulated by neuronal activity. Our approach greatly facilitates investigations of actin dynamics at the nanoscale within functionally intact brain tissue.

Binding Kinetics of Calbindin-D28k Determined by Flash Photolysis of Caged Ca2+

We have used UV flash photolysis of DM-nitrophen in combination with model-based analysis of Oregon Green 488 BAPTA-5N fluorescence transients to study the kinetics of Ca(2+) binding to calbindin-D(28K). The experiments used saturated DM-nitrophen at a [Ca(2+)] of 1.5 microM. Under these conditions, UV laser flashes produced rapid steplike increases in [Ca(2+)] in the absence of calbindin-D(28K), and in its presence the decay of the flash-induced fluorescence was due solely to the Ca(2+) buffering by the protein. We developed a novel method for kinetic parameter derivation and used the synthetic Ca(2+) buffer EGTA to confirm its validity. We provide evidence that calbindin-D(28K) binds Ca(2+) in at least two distinct kinetic patterns, one arising from high-affinity sites that bind Ca(2+) with a k(on) comparable to that of EGTA (i.e., approximately 1 x 10(7) M(-1) s(-1)) and another with lower affinity and an approximately eightfold faster k(on). In view of the inability of conventional approaches to adequately resolve rapid Ca(2+) binding kinetics of Ca(2+) buffers, this method promises to be highly valuable for studying the Ca(2+) binding properties of other biologically important Ca(2+) binding proteins.

Two-Color STED Microscopy of Living Synapses Using A Single Laser-Beam Pair

The advent of superresolution microscopy has opened up new research opportunities into dynamic processes at the nanoscale inside living biological specimens. This is particularly true for synapses, which are very small, highly dynamic, and embedded in brain tissue. Stimulated emission depletion (STED) microscopy, a recently developed laser-scanning technique, has been shown to be well suited for imaging living synapses in brain slices using yellow fluorescent protein as a single label. However, it would be highly desirable to be able to image presynaptic boutons and postsynaptic spines, which together form synapses, using two different fluorophores. As STED microscopy uses separate laser beams for fluorescence excitation and quenching, incorporation of multicolor imaging for STED is more difficult than for conventional light microscopy. Although two-color schemes exist for STED microscopy, these approaches have several drawbacks due to their complexity, cost, and incompatibility with common labeling strategies and fluorophores. Therefore, we set out to develop a straightforward method for two-color STED microscopy that permits the use of popular green-yellow fluorescent labels such as green fluorescent protein, yellow fluorescent protein, Alexa Fluor 488, and calcein green. Our new (to our knowledge) method is based on a single-excitation/STED laser-beam pair to simultaneously excite and quench pairs of these fluorophores, whose signals can be separated by spectral detection and linear unmixing. We illustrate the potential of this approach by two-color superresolution time-lapse imaging of axonal boutons and dendritic spines in living organotypic brain slices.

Two-Photon Excitation STED Microscopy in Two Colors in Acute Brain Slices

Many cellular structures and organelles are too small to be properly resolved by conventional light microscopy. This is particularly true for dendritic spines and glial processes, which are very small, dynamic, and embedded in dense tissue, making it difficult to image them under realistic experimental conditions. Two-photon microscopy is currently the method of choice for imaging in thick living tissue preparations, both in acute brain slices and in vivo. However, the spatial resolution of a two-photon microscope, which is limited to ~350 nm by the diffraction of light, is not sufficient for resolving many important details of neural morphology, such as the width of spine necks or thin glial processes. Recently developed superresolution approaches, such as stimulated emission depletion microscopy, have set new standards of optical resolution in imaging living tissue. However, the important goal of superresolution imaging with significant subdiffraction resolution has not yet been accomplished in acute brain slices. To overcome this limitation, we have developed a new microscope based on two-photon excitation and pulsed stimulated emission depletion microscopy, which provides unprecedented spatial resolution and excellent experimental access in acute brain slices using a long-working distance objective. The new microscope improves on the spatial resolution of a regular two-photon microscope by a factor of four to six, and it is compatible with time-lapse and simultaneous two-color superresolution imaging in living cells. We demonstrate the potential of this nanoscopy approach for brain slice physiology by imaging the morphology of dendritic spines and microglial cells well below the surface of acute brain slices.

Early synaptic deficits in the APP/PS1 mouse model of Alzheimer's disease involve neuronal adenosine A2A receptors.

Synaptic plasticity in the autoassociative network of recurrent connections among hippocampal CA3 pyramidal cells is thought to enable the storage of episodic memory. Impaired episodic memory is an early manifestation of cognitive deficits in Alzheimers disease (AD). In the APP/PS1 mouse model of AD amyloidosis, we show that associative long-term synaptic potentiation (LTP) is abolished in CA3 pyramidal cells at an early stage. This is caused by activation of upregulated neuronal adenosine A2A receptors (A2AR) rather than by dysregulation of NMDAR signalling or altered dendritic spine morphology. Neutralization of A2AR by acute pharmacological inhibition, or downregulation driven by shRNA interference in a single postsynaptic neuron restore associative CA3 LTP. Accordingly, treatment with A2AR antagonists reverts one-trial memory deficits. These results provide mechanistic support to encourage testing the therapeutic efficacy of A2AR antagonists in early AD patients.

Calcium‐dependent inactivation of high‐threshold calcium currents in human dentate gyrus granule cells

1 Dentate gyrus granule cells acutely dissociated from hippocampal slices obtained from chronic temporal lobe epilepsy (TLE) patients displayed a high‐voltage activated (HVA) Ca2+ conductance with a pronounced Ca2+‐dependent inactivation. 2 Inactivation time constants and peak HVA Ca2+ current (ICa) amplitudes did not differ between perforated patch and whole‐cell recordings without added exogenous Ca2+ buffers, indicating that the Ca2+‐dependent characteristics of ICa inactivation were well preserved in whole‐cell recordings. 3 Inactivation time constants correlated with whole‐cell ICa, and were increased when Ca2+ was replaced with Ba2+ in the external solution or 5 mm BAPTA was added to the pipette solution. 4 In recordings without added exogenous Ca2+ buffers, the time course of ICa inactivation was comparable between human TLE and kindled rat granule cells. Conversely, the time course of ICa in human TLE granule cells loaded with 5 mm intracellular BAPTA resembled that observed in buffer‐free recordings from control rat neurones. 5 The loss of a putative intraneuronal Ca2+ buffer, the Ca2+‐binding protein calbindin (CB), from human granule cells during TLE may result in the pronounced Ca2+‐dependent ICa inactivation. This process could serve a neuroprotective role by significantly decreasing Ca2+ entry during prolonged trains of action potentials known to occur during seizures.