When hippocampal synapses in culture are pharmacologically silenced for several days, synaptic strength increases. The structural correlate of this change in strength is an increase in the size of the synapses, with all synaptic components--active zone, postsynaptic density, and bouton--becoming larger. Further, the number of docked vesicles and the total number of vesicles per synapse increases, although the number of docked vesicles per area of active zone is unchanged. In parallel with these anatomical changes, the physiologically measured size of the readily releasable pool (RRP) and the release probability are increased. Ultrastructural analysis of individual synapses in which the RRP was previously measured reveals that, within measurement error, the same number of vesicles are docked as are estimated to be in the RRP.

Genetically encoded sensors of glutamate concentration are based on FRET between cyan and yellow fluorescent proteins bracketing a bacterial glutamate-binding protein. Such sensors have yet to find quantitative applications in neurons, because of poor response amplitude in physiological buffers or when expressed on the neuronal cell surface. We have improved our glutamatesensing fluorescent reporter (GluSnFR) by systematic optimization of linker sequences and glutamate affinities. Using SuperGluSnFR, which exhibits a 6.2-fold increase in response magnitude over the original GluSnFR, we demonstrate quantitative optical measurements of the time course of synaptic glutamate release, spillover, and reuptake in cultured hippocampal neurons with centisecond temporal and spine-sized spatial resolution. During burst firing, functionally significant spillover persists for hundreds of milliseconds. These glutamate levels appear sufficient to prime NMDA receptors, potentially affecting dendritic spike initiation and computation. Stimulation frequency-dependent modulation of spillover suggests a mechanism for nonsynaptic neuronal communication.fluorescence resonance energy transfer ͉ hippocampal neurons ͉ synaptic release G lutamate is the primary excitatory neurotransmitter in the brain, and precise measurement of its spatiotemporal pattern of synaptic release and propagation would provide insight into diverse brain processes, including synaptic crosstalk, cerebral ischemia, and mechanisms of learning and memory. In hippocampal slices, synaptic glutamate spillover to the dendrite and neighboring synapses induces homeostatic regulation of glutamate release through extrasynaptic mGluR activation (1), limits synaptic independence (2), lengthens EPSC durations (3), and permits heterosynaptic LTP/LTD (4). Spillover is a primary means of chemical neurotransmission between mitral cells in the rat olfactory bulb (5) and between climbing fibers and molecular layer interneurons in cerebral cortex (6). Estimates of glutamate concentration and dynamics in synaptic, extrasynaptic, and extracellular compartments have been made by NMDAR antagonist displacement (7), glutamate uptake inhibitor application (2), whole ''sniffer'' cells (8), outside-out ''sniffer'' patch electrodes (9, 10), patch recording of astrocyte synaptically evoked transporter currents (STCs) (11), enzymatically coupled electrochemical probes (12), enzymatically coupled metabolite imaging (13), and other methods. Although each method provided a new perspective on glutamate action, all were hampered by a lack of resolution in the spatial or temporal domains because of single-site measurement, reliance on partially coupled or confounded currents, desensitizing receptors, or indirect and slow secondary cascades.Recently, the glutamate reporters glutamate-sensing fluorescent reporter (GluSnFR) ʈ (14) and fluorescent indicator protein for glutamate (FLIPE) (15) were constructed by linear genetic fusions of the glutamate periplasmic binding protein GltI (also known as ybeJ) ...

The mechanisms that contribute to the extinction of previously acquired memories are not well understood. These processes, often referred to as inhibitory learning, are thought to be parallel learning mechanisms that require a reacquisition of new information and suppression of previously acquired experiences in order to adapt to novel situations. Using newly generated metabotropic glutamate receptor 5 (mGluR5) knock-out mice, we investigated the role of mGluR5 in the acquisition and reversal of an associative conditioned task and a spatial reference task. We found that acquisition of fear conditioning is partially impaired in mice lacking mGluR5. More markedly, we found that extinction of both contextual and auditory fear was completely abolished in mGluR5 knock-out mice. In the Morris Water Maze test (MWM), mGluR5 knock-out mice exhibited mild deficits in the rate of acquisition of the regular water maze task, but again had significant deficits in the reversal task, despite overall spatial memory being intact. Together, these results demonstrate that mGluR5 is critical to the function of neural circuits that are required for inhibitory learning mechanisms, and suggest that targeting metabotropic receptors may be useful in treating psychiatric disorders in which aversive memories are inappropriately retained.

Summary Synaptic vesicle recycling is essential for maintaining efficient synaptic transmission. Detailed dissection of single vesicle recycling still remains a major challenge. We have developed a new fluorescent pH reporter that permits us to follow the fate of individual vesicles at hippocampal synapses after exocytosis. Here we show that, during low frequency stimulation, single vesicle fusion leads to two distinct vesicle internalizations, instead of one, as in general perception: one by a fast endocytosis pathway (~3s), the other by a slow endocytosis pathway (after 10s). The exocytosed vesicular proteins are preferentially recaptured in both pathways. RNAi knocking down of clathrin inhibits both pathways. As stimulation frequency increases, the number of endocytosed vesicles begins to match antecedent exocytosis. Meanwhile, the slow endocytosis is accelerated and becomes the predominant pathway. These results reveal that two pathways of endocytosis are orchestrated during neuronal activity, enabling the highly efficient endocytosis machinery at central synapses.