Dorsal commissural axons in the developing spinal cord cross the floor plate, then turn rostrally and grow along the longitudinal axis, close to the floor plate. We used a subtractive hybridization approach to identify guidance cues responsible for the rostral turn in chicken embryos. One of the candidates was the morphogen Sonic hedgehog (Shh). Silencing of the gene SHH (which encodes Shh) by in ovo RNAi during commissural axon navigation demonstrated a repulsive role in post-commissural axon guidance. This effect of Shh was not mediated by Patched (Ptc) and Smoothened (Smo), the receptors that mediate effects of Shh in morphogenesis and commissural axon growth toward the floor plate. Rather, functional in vivo studies showed that the repulsive effect of Shh on postcommissural axons was mediated by Hedgehog interacting protein (Hip).

Synaptic plasticity is considered essential for learning and storage of new memories. Whether all synapses on a given neuron have the same ability to express long-term plasticity is not well understood. Synaptic microanatomy could affect the function of local signaling cascades and thus differentially regulate the potential for plasticity at individual synapses. Here, we investigate how the presence of endoplasmic reticulum (ER) in dendritic spines of CA1 pyramidal neurons affects postsynaptic signaling. We show that the ER is targeted selectively to large spines containing strong synapses. In ER-containing spines, we frequently observed synaptically triggered calcium release events of very large amplitudes. Low-frequency stimulation of these spines induced a permanent depression of synaptic potency that was independent of NMDA receptor activation and specific to the stimulated synapses. In contrast, no functional changes were induced in the majority of spines lacking ER. Both calcium release events and long-term depression depended on the activation of metabotropic glutamate receptors and inositol trisphosphate receptors. In summary, spine microanatomy is a reliable indicator for the presence of specific signaling cascades that govern plasticity on a micrometer scale.long-term depression ͉ metabotropic glutamate receptor ͉ metaplasticity ͉ spine apparatus ͉ dendritic spines A ctivity-dependent changes in synaptic strength are thought to be essential for learning and the formation of new memories (1). The intracellular signaling cascades underlying different forms of synaptic plasticity have been studied extensively at the CA3 to CA1 projection in the hippocampus. Although long-term potentiation at these synapses is strictly NMDA receptor-dependent, at least two mechanistically distinct forms of long-term depression (LTD) have been described, triggered by the activation of NMDA receptors (NMDARs) and metabotropic glutamate receptors (mGluRs), respectively (2). Although the potential for NMDAR-dependent plasticity can be regulated by the subunit composition of the receptor itself, much less is known about the regulation of mGluR-dependent plasticity (3). Aberrant mGluR signaling and dysregulated synaptic plasticity have been implicated in severe mental disorders, such as fragile X mental retardation (4). The induction of mGluRdependent LTD is known to involve activation of postsynaptic group I mGluRs and inositol trisphosphate (IP 3 )-mediated calcium release from the endoplasmic reticulum (ER) (reviewed in ref. 5). Interestingly, only a small subset of dendritic spines on CA1 pyramidal cells contains ER (6). The heterogeneous distribution of this organelle very well could affect the plasticity of individual synapses (7,8).In all previous studies of synaptic depression, plasticity was induced at large numbers of synapses simultaneously. However, this strategy does not allow the investigation of functional differences between individual synaptic connections. Differences in synaptic microanatomy, such as the presen...

Dendritic spines have been proposed to function as electrical compartments for the active processing of local synaptic signals. However, estimates of the resistance between the spine head and the parent dendrite suggest that compartmentalization is not tight enough to electrically decouple the synapse. Here we show in acute hippocampal slices that spine compartmentalization is initially very weak, but increases dramatically upon postsynaptic depolarization. Using NMDA receptors as voltage sensors, we provide evidence that spine necks not only regulate diffusional coupling between spines and dendrites, but also control local depolarization of the spine head. In spines with high-resistance necks, presynaptic activity alone was sufficient to trigger calcium influx through NMDA receptors and R-type calcium channels. We conclude that calcium influx into spines, a key trigger for synaptic plasticity, is dynamically regulated by spine neck plasticity through a process of electrical compartmentalization.

Spike timing-dependent long-term potentiation (t-LTP) is the embodiment of Donald Hebb's postulated rule for associative memory formation. Pre-and postsynaptic action potentials need to be precisely correlated in time to induce this form of synaptic plasticity. NMDA receptors have been proposed to detect correlated activity and to trigger synaptic plasticity. However, the slow kinetic of NMDA receptor currents is at odds with the millisecond precision of coincidence detection. Here we show that AMPA receptors are responsible for the extremely narrow time window for t-LTP induction. Furthermore, we visualized synergistic interactions between AMPA and NMDA receptors and back-propagating action potentials on the level of individual spines. Supralinear calcium signals were observed for spike timings that induced t-LTP and were most pronounced in spines well isolated from the dendrite. We conclude that AMPA receptors gate the induction of associative synaptic plasticity by regulating the temporal precision of coincidence detection.orrelated activity in connected neurons can trigger longlasting changes in synaptic strength, in which sign and magnitude of synaptic modifications depend on the relative timing of pre-and postsynaptic action potentials (1-3). Presynaptic activity followed by postsynaptic action potentials generally leads to an increase in synaptic strength (timing-dependent long-term potentiation, t-LTP), whereas activity in the reverse order induces long-term depression. Remarkably, the existence of t-LTP was predicted >60 y ago by the Canadian psychologist Donald Hebb as a mechanism for associative learning (4). Although t-LTP is considered a crucial mechanism for activity-dependent modifications of brain circuits, the biophysics of coincidence detection are not fully understood. The required coincidence detector needs to measure the relative timing of postsynaptic action potentials (APs) with respect to the brief glutamate transient in the synaptic cleft with millisecond precision and to convert this temporal measurement into a synapse-specific biochemical signal. Postsynaptic NMDA receptors (NMDARs), due to their sensitivity to both glutamate and membrane depolarization, have been proposed to act as detectors of temporal coincidence. Ca 2+ influx through NMDARs activates a series of biochemical processes that eventually lead to strengthening of the synaptic connection (5-9). However, there is a striking mismatch between the slow kinetics of NMDARs and the very brief time window in which t-LTP can be induced, suggesting that an additional mechanism is necessary to sharpen the timing sensitivity (10-12).Here we investigate the role of AMPA receptors (AMPARs) during coincidence detection at Schaffer collateral synapses. Modulation of AMPAR currents during coincident activity strongly affected the induction of synaptic plasticity by pairing of preand postsynaptic spikes. Furthermore, we visualized NMDARdependent calcium signals in individual spines of CA1 pyramidal cells. During pairing stimulation, AMPAR ...

Over the past few years, the light-gated cation channel Channelrhodopsin-2 (ChR2) has seen a remarkable diversity of applications in neuroscience. However, commonly used wide-field illumination provides poor spatial selectivity for cell stimulation. We explored the potential of focal laser illumination to map photocurrents of individual neurons in sparsely transfected hippocampal slice cultures. Interestingly, the best spatial resolution of photocurrent induction was obtained at the lowest laser power. By adjusting the light intensity to a neuron's spike threshold, we were able to trigger action potentials with a spatial selectivity of less than 30 lm. Experiments with dissociated hippocampal cells suggested that the main factor limiting the spatial resolution was ChR2 current density rather than scattering of the excitation light. We conclude that subcellular resolution can be achieved only in cells with a high ChR2 expression level and that future improved variants of ChR2 are likely to extend the spatial resolution of photocurrent induction to the level of single dendrites.

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