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Dendritic spines receive most excitatory inputs in the vertebrate brain, but their function is still poorly understood. Using two-photon calcium imaging of CA1 pyramidal neurons in rat hippocampal slices, we investigated the mechanisms by which calcium enters into individual spines in the stratum radiatum. We find three different pathways for calcium influx: high-threshold voltage-sensitive calcium channels, NMDA receptors, and an APV-resistant influx consistent with calcium-permeable AMPA or kainate receptors. These pathways vary among different populations of spines and are engaged under different stimulation conditions, with peak calcium concentrations reaching >10 microM. Furthermore, as a result of the biophysical properties of the NMDA receptor, the calcium dynamics of spines are exquisitely sensitive to the temporal coincidence of the input and output of the neuron. Our results confirm that individual spines are chemical compartments that can perform coincidence detection. Finally, we demonstrate that functional studies and optical quantal analysis of single, identified synapses is feasible in mammalian CNS neurons in brain slices.

Dendritic spines receive most excitatory inputs in the CNS and compartmentalize calcium. Although the mechanisms of calcium influx into spines have been explored, it is unknown what determines the calcium decay kinetics in spines. With two-photon microscopy we investigate action potential-induced calcium dynamics in spines from rat CA1 pyramidal neurons in slices. The [Ca(2+)](i) in most spines shows two decay kinetics: an initial fast component, during which [Ca(2+)](i) in spines decays to dendritic levels, followed by a slower decay phase in which the spine follows dendritic kinetics. The correlation between [Ca(2+)](i) in spine and dendrite at the breakpoint of the decay kinetics demonstrates diffusional equilibration between spine and dendrite during the slower component. To explain the faster initial decay, we rule out saturation or kinetic effects of endogenous or exogenous buffers and focus instead on (1) active calcium extrusion and (2) buffered diffusion of calcium from spine to dendrite. The presence of an undershoot in most spines indicates that extrusion mechanisms can be intrinsic to the spine. Supporting the two mechanisms, pharmacological blockade of smooth endoplasmic reticulum calcium (SERCA) pumps and the length of the spine neck affect spine decay kinetics. Using a mathematical model, we find that the contribution of calcium pumps and diffusion varies from spine to spine. We conclude that dendritic spines have calcium pumps and that their density and kinetics, together with the morphology of the spine neck, determine the time during which the spine compartmentalizes calcium.

Dendritic spines receive most excitatory inputs in the CNS and compartmentalize calcium. Spines also undergo rapid morphological changes, although the function of this motility is still unclear. We have investigated the effect of spine movement on spine calcium dynamics with two-photon photobleaching of enhanced green fluorescent protein and calcium imaging of action potential-elicited transients in spines from layer 2/3 pyramidal neurons in mouse visual cortex slices. The elongation or retraction of the spine neck during spine motility alters the diffusional coupling between spine and dendrite and significantly changes calcium decay kinetics in spines. Our results demonstrate that the spine's ability to compartmentalize calcium is constantly changing. Key words: GFP; imaging; two photon; photobleaching; LTP; neocortexAs first predicted by Ramón y Cajal (1891), dendritic spines receive most synaptic inputs in the mammalian CNS (Gray, 1959;Harris and Kater, 1994). Spines are separated from their parent dendrites by a thin neck and compartmentalize calcium during synaptic stimulation (Müller and Connor, 1991;Yuste and Denk, 1995;Yuste et al., 2000). Calcium compartmentalization in spines is likely to be functionally important, because calcium mediates input-specific forms of synaptic plasticity (Lynch et al., 1983;Malenka et al., 1989). Calcium decay kinetics in spines is controlled by diffusion of calcium across the spine neck and active removal of calcium from the spine cytoplasm (Majewska et al., 2000a) as well as by calcium buffers endogenous to the spine head. Therefore, the morphology of the spine neck and the expression and regulation of calcium pumps and buffers control the duration of calcium transients in spines.Spines have been shown recently to be extremely motile on the timescale of seconds (Fischer et al., 1998;Dunaevsky et al., 1999). The function of spine motility is still unclear. Although new filopodia and spines can appear after electrical stimulation (Engert and Bonhoeffer, 1999;Maletic-Savatic et al., 1999), basal motility, such as that present in the absence of stimulation, appears resilient to major changes in the activity of the cell (Dunaevsky et al., 1999). Both spine and filopodial motility declines with development (Dailey and Smith, 1996;Ziv and Smith, 1996;Dunaevsky et al., 1999) and is thought to be related to critical periods of synaptic rearrangements (Dunaevsky et al., 1999;Lendvai et al., 2000). Finally, volatile anesthetics block spine motility, suggesting that rapid motility may play a global role in brain function (Kaech et al., 1999).The finding that calcium decays in spines are regulated by calcium diffusion through the spine neck (Majewska et al., 2000a) suggests that spine motility could alter this diffusional coupling and potentially modify spine calcium decay kinetics. On the other hand, calcium pumps at the spine (Majewska et al., 2000a) and endogenous calcium buffers also control decay kinetics, and the type and amount of motility could be too small to produce signific...

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