Florian Engert

Dendritic spine changes associated with hippocampal long-term synaptic plasticity.

Long-term enhancement of synaptic efficacy in the hippocampus is an important model for studying the cellular mechanisms of neuronal plasticity, circuit reorganization, and even learning and memory. Although these long-lasting functional changes are easy to induce, it has been very difficult to demonstrate that they are accompanied or even caused by morphological changes on the subcellular level. Here we combined a local superfusion technique, with two-photon imaging, which allowed us to scrutinize specific regions of the postsynaptic dendrite where we knew that the synaptic changes had to occur. We show that after induction of long-lasting (but not short-lasting) functional enhancement of synapses in area CA1, new spines appear on the postsynaptic dendrite, whereas in control regions on the same dendrite or in slices where long-term potentiation was blocked, no significant spine growth occurred.

Synapse specificity of long-term potentiation breaks down at short distances

Long-term potentiation (LTP), the long-lasting increase in synaptic transmission, has been proposed to be a cellular mechanism essential for learning and memory, neuronal development, andcircuit reorganization. In the original theoretical and experimental work it was assumed that only synapses that had experienced concurrent pre- and postsynaptic activity are subject to synaptic modification. It has since been shown, however, that LTP is also expressed in synapses on neighbouring neurons that have not undergone the induction procedure. Yet, it is still believed that this spread of LTP is limited to adjacent postsynaptic cells, and does not occur for synapses on neighbouring input fibres,,. However, for technical reasons, tests for ‘input specificity’ were always done for synapses relatively far apart. Here we have used a new local superfusion technique, which allowed us to assess the synaptic specificity of LTP with a spatial resolution of ∼30μm. Our results indicate that there is no input specificity at a distance of less than 70μm. Synapses in close proximity to a site of potentiation are also potentiated regardless of their own history of activation, whereas synapses far away show no potentiation.

Moving visual stimuli rapidly induce direction sensitivity of developing tectal neurons

During development of the visual system, the pattern of visual inputs may have an instructive role in refining developing neural circuits. How visual inputs of specific spatiotemporal patterns shape the circuit development remains largely unknown. We report here that, in the developing Xenopus retinotectal system, the receptive field of tectal neurons can be ‘trained’ to become direction-sensitive within minutes after repetitive exposure of the retina to moving bars in a particular direction. The induction of direction-sensitivity depends on the speed of the moving bar, can not be induced by random visual stimuli, and is accompanied by an asymmetric modification of the tectal neurons receptive field. Furthermore, such training-induced changes require spiking of the tectal neuron and activation of a NMDA (N-methyl-d-aspartate) subtype of glutamate receptors during training, and are attributable to an activity-induced enhancement of glutamate-mediated inputs. Thus, developing neural circuits can be modified rapidly and specifically by visual inputs of defined spatiotemporal patterns, in a manner consistent with predictions based on spike-time-dependent synaptic modification.

A fast activating presynaptic reuptake current during serotonergic transmission in identified neurons of Hirudo

An electrogenic serotonin (5-HT) uptake process was characterized in the serotonergic Retzius-P cell synapse of the leech, and the simultaneous activation of this presynaptic reuptake and the postsynaptic response was monitored during evoked transmitter release. A presynaptic, Na(+)-dependent inward current upon application of 5-HT was isolated at membrane potentials between -80 and +60 mV. Its identification as a transmitter uptake current was confirmed by monitoring accumulation of the autofluorescent 5-HT analog 5,7-dihydroxytryptamine during activation of this current. To study the kinetics of 5-HT reuptake in functional synapses, transmitter release was stimulated by flash photolysis of the Ca(2+)-caging DM-nitrophen. The results demonstrate that reuptake activates with a minimal delay of less than a millisecond during synaptic transmission. It acts as a rapid transmitter removal system to determine the time course of the postsynaptic response and monitors the kinetics of transmitter clearance at the synaptic site.

Emergence of Input Specificity of LTP during Development of Retinotectal Connections In Vivo

Input specificity of activity-induced synaptic modification was examined in the developing Xenopus retinotectal connections. Early in development, long-term potentiation (LTP) induced by theta burst stimulation (TBS) at one retinal input spreads to other unstimulated converging inputs on the same tectal neuron. As the animal develops, LTP induced by the same TBS becomes input specific, a change that correlates with the increased complexity of tectal dendrites and more restricted distribution of dendritic Ca(2+) evoked by each retinal input. In contrast, LTP induced by 1 Hz correlated pre- and postsynaptic spiking is input specific throughout the same developmental period. Thus, input specificity of LTP emerges with neural development and depends on the pattern of synaptic activity.

FAST LOCAL SUPERFUSION TECHNIQUE

A system for rapid, local superfusion of cultured neurones and their neurites with various different test drugs is elucidated. An area of down to 30 μm diameter was superfused with the aid of two micropipettes, one for delivering the test solution and the other for its removal. Active removal of solution within the dead-space of the delivery pipette guarantees, on the one hand, fast and flexible pressure control and, on the other, enables the quick exchange (<1 s) of multiple solutions. By increasing the pressure in the superfusion pipette, the laminar stream between the pipettes was forced down onto the cell layer. The change from bath to superfusion solutions, evaluated by liquid junction potential changes, occurs in the order of 1 ms.

A low-cost UV laser for flash photolysis of caged compounds

Photolysis of caged compounds has become a standard tool for the rapid application of bioactive molecules. In principle this technique also allows to apply substances in a spatially very restricted manner. An important practical limitation for such experiments, however, is the high cost of UV lasers. Here we describe the assembly of an inexpensive pulsed nitrogen laser which is suitable for photolysis experiments. The laser which can be constructed in less than 1 week and for less than US

Different effects of baclofen and GTPγS on voltage-activated Ca2+ currents in rat hippocampal neurons in vitro

500 emits light pulses with a duration of approximately 5 ns, an energy of up to 200 microJ (= 40 kW) and a wavelength of 337 nm. Its beam can be focused to roughly 30 microns, a firing frequency of up to 50 Hz can be achieved, and electrical artifacts are minimal. These specifications make the laser optimally suited for most photolysis experiments. Its low price and ease of use should make the technique of spatially restricted flash photolysis amenable to many laboratories.

The assembly of neural circuits.

Reduction of voltage-activated Ca2+ currents by intracellular application of guanosine 5-O-(3-thiotriphosphate) (GTP gamma S) through ultraviolet (UV) photolysis of the caged compound, is followed by a re-augmentation to control levels within 10 min, independently of the divalent cation used. The Ca2+ current inhibition by the gamma-aminobutyric acid type B (GABAB) receptor agonist baclofen, which is also thought to be mediated by a GTP-binding protein (G-protein), is potentiated when GTP gamma S is uncaged during agonist superfusion. The authors suggest that GTP gamma S activates G-protein-dependent pathways that are not activated by the baclofen receptor.

Verschwommene Erinnerungen - Synaptische Verstärkung und ihre lokalen Effekte

when engaged by EphA4, suggesting that proteolysis and endocytosis may function in parallel. These studies Cambridge, Massachusetts 02138 3 Department of Molecular and Cellular Biology open up an interesting question of whether endocytosis is simply involved in detachment or may instead participate actively as part of a signaling mechanism. Endocy-tosis may not be a mechanism exclusive for the ephrins. In fact, endocytosis has been shown to occur during The enormous complexity and the incredible precision Sema3A-mediated growth cone collapse (Fournier et al., 2000; Jurney et al., 2002). Christine Holt of Cambridge of neuronal connectivity have fascinated researchers for over a century. This past fall, Lago di Como was the University presented data to show that reverse-direction endocytosis occurs in retinal growth cones when site of a gathering of a diverse group of neuroscientists at the Assembly of Neural Circuits meeting, organized EphB2-Fc is presented to ephrin-B1-expressing retinal cells and that endocytosis is functionally required for Tamagnone and sponsored by EMBO. This meeting growth cone collapse. This reverse endocytosis is a rapid process that is only triggered by unclustered EphB2 ecto-highlighted recent findings on the cellular mechanisms of axon guidance, growth cone remodeling, new axon domains and requires proteosome function (Mann et al., 2003). Interestingly, forward signaling that leads to guidance cues, mechanisms of in vivo guidance events, specificity of synaptic connections, and the role of neu-growth cone collapse appears to require clustered ephrin-B1 ectodomain and is proteosome independent. ral activity in circuit development. Endocytosis was not detected in the forward direction. These results illustrate interesting differences in ephrin/ 1. Cell Biology of Growth Cone Guidance Progress over recent years has endowed us with the Eph signaling systems in two directions. Although the functional consequences of these distinctions are not knowledge of a large number of axon guidance molecules and their cognate receptors, and in many cases, yet clear, these differences may provide an opportunity to allow regulatory signals to exert specific or different the downstream signaling components have also been identified. For the most part, however, the specific cellu-influences on the two partners, leading to different downstream events in the two cells that are in contact. lar mechanisms that mediate guidance remain mysterious. Membrane Compartmentalization Endocytosis and Growth Cone Guidance In addition to being expressed in the surrounding envi-Several talks focused on the role of endocytosis in ronment, guidance cues are also frequently found ex-growth cone guidance. For …